key: cord-0736700-updjkr8w
authors: Tundo, G.R.; Sbardella, D.; Santoro, A.M.; Coletta, A.; Oddone, F.; Grasso, G.; Milardi, D.; Lacal, P.; Marini, S.; Purrello, P.; Graziani, G.; Coletta, M.
title: The proteasome as a druggable target with multiple therapeutic potentialities: Cutting and non-cutting edges
date: 2020-05-19
journal: Pharmacol Ther
DOI: 10.1016/j.pharmthera.2020.107579
sha: c6ca840f6c446c66396c06fc42401af987b88e5d
doc_id: 736700
cord_uid: updjkr8w

Ubiquitin Proteasome System (UPS) is an adaptable and finely tuned system that sustains proteostasis network under a large variety of physiopathological conditions. Its dysregulation is often associated with the onset and progression of human diseases; hence, UPS modulation has emerged as a promising new avenue for the development of treatments of several relevant pathologies, such as cancer and neurodegeneration. The clinical interest in proteasome inhibition has considerably increased after the FDA approval in 2003 of bortezomib for relapsed/refractory multiple myeloma, which is now used in the front-line setting. Thereafter, two other proteasome inhibitors (carfilzomib and ixazomib), designed to overcome resistance to bortezomib, have been approved for treatment-experienced patients, and a variety of novel inhibitors are currently under preclinical and clinical investigation not only for haematological malignancies but also for solid tumours. However, since UPS collapse leads to toxic misfolded proteins accumulation, proteasome is attracting even more interest as a target for the care of neurodegenerative diseases, which are sustained by UPS impairment. Thus, conceptually, proteasome activation represents an innovative and largely unexplored target for drug development. According to a multidisciplinary approach, spanning from chemistry, biochemistry, molecular biology to pharmacology, this review will summarize the most recent available literature regarding different aspects of proteasome biology, focusing on structure, function and regulation of proteasome in physiological and pathological processes, mostly cancer and neurodegenerative diseases, connecting biochemical features and clinical studies of proteasome targeting drugs.

intracellular proteolytic systems, namely Ubiquitin-Proteasome-System (UPS) and autophagy (Klaips et al., 2018; Ciechanover and Kwon, 2017; Sala et al., 2017) . Furthermore, a myriad of regulatory proteins (such as transcription and metabolic factors, chromatin remodelling factors, and regulators of posttranslational modifications) act as PN auxiliary and coordinate the cross-talk between the PN compartments accounting for the afore mentioned plasticity of the PN (Labbadia and Morimoto, 2015; Klaips et al., 2018) . Therefore, unlike early scientists, who considered proteins essentially stable and prone to only a minor "wear and tear" (Schoenheimer et al., 1939; Schoenheimer, 1942; Thibaudeau and Smith, 2019) , it is now known that proteome is highly dynamic, and proteins constantly undergo turn over at different rates, according to their biological role (Lecker et al., 2006; Thibaudeau and Smith, 2019) .

In the 1950s, the discovery of autophagy-lysosome system as "intracellular exergonic digestive system" by de Duve and colleagues was the first step in understanding intracellular and extracellular protein breakdown (de Duve et al., 1953; de Duve et al., 1955; de Duve and Wattiaux, 1966; Sabatini and Adesnik, 2013) . Over the same years, Simpson showed for the first time that intracellular proteolysis in mammalian cells requires energy, suggesting the existence of an additional mechanism of protein degradation (Simpson et al., 1953) . However, this observation was considered with scepticism, since hydrolysis of the peptide bond is exergonic, and there is no apparent thermodynamic advantage in energy use (Wilkinson et al., 2005) . However, the seminal Simpson's discovery found support in the 1970s, when Goldberg and colleagues identified a novel, cytosolic ATP-dependent proteolytic system (Goldberg, 1972; Goldberg and Dice, 1974; Goldberg and St John, 1974; Etlinger and Goldberg, 1977; Bigelow et al., 1981; Wilkinson et al., 2005; Thibaudeau and Smith, 2019) . Some years later, Wilk and Orlowski purified a 700-kDa "multicatalytic proteinase complex", which was able to cleave peptides after hydrophobic, acidic and basic residues, suggesting the existence of multiple active sites in its structure (Wilk and Orlowski, 1980; Wilk and Orlowski, 1983) . This "stacked donut ring" complex (which later was shown to be the 20S) was tnamed "proteasome", and its orthologues were identified in all life domains (Tanaka et al., 1983; Tanaka et al., 1988; Arrigo et al., 1998; Thibaudeau and Smith, 2019) . A milestone in protein degradation field was the discovery by Ciechanover and colleagues of a 8-kDa heat-stable protein, APF-1 (later renamed "ubiquitin"), whose ATP-dependent covalent conjugation with proteins targeted them for degradation by a downstream protease, that was then identified as the 26S proteasome (Ciechanover et al., 1978; Ciechanover et al., 1980; Ciechanover et al., 1982; Hershko et al., 1980; Hershko et al., 1982; Ciechanover et al., 1984; Hough et al., 1986;  J o u r n a l P r e -p r o o f Journal Pre-proof specificity (Hough et al., 1986; Pickart et al., 2001; Glickman and Ciechanover, 2002; Windheim et al., 2008; Ciechanover, 2013; Leestemaker and Ovaa, 2017; Pao et al 2018) . The end-point of UPS is the 26S complex (hereafter referred to as 26S), a multifunctional 2500 kDa proteolytic molecular machine, composed by the 20S proteasome core particle (CP, hereafter referred to as 20S), which houses the proteolytic activity. The 20S is capped by one or two 19S regulatory particle(s) (RP) (hereafter referred to as 19S), which carry out the ATP-dependent recognition, unfolding and translocation into the 20S of the poly-ubiquitinated substrate (Ciechanover, 2015; Glickman and Ciechanover, 2002; Kunjappu and Hochstrasser, 2014; Pao et al., 2018 , see also sections 2.2 and 2.3). Over the last decades, several alternative regulators of 20S have been described, namely PA28 protein family and Blm10/PA200, whose structure, substrate specificities, and biological roles go beyond the scope of this review and are extensively reviewed elsewhere (Rechsteiner and Hill, 2005; Tanaka et al., 2009; Huang and Chen. 2009; Kish-Trier, 2013; Schmidt and Finley, 2014; Jiang et al., 2018; Limanaqi et al., 2019) .

Although the initial dogma on proteasome recognition mechanism states that the 26S hydrolyses only proteins tagged with at least four ubiquitin molecules, emerging evidences show that polyubiquitin chains are not the unique signal. In fact, in some cases, multiple or single monoubiquitination appears to be sufficient to label a substrate for proteasomal degradation (Shabek, 2012; Kravtsova-Ivantsiv, 2009 ). Moreover, ornithine decarboxylase has been the first of a long series of protein substrates (i.e., Rpn4, thymidylate synthase, myelin) reported to be degraded by the 26S regardless of ubiquitination (Rosenberg-Hasson et al., 1989; Bercovich et al., 1989; Murakami et al., 1992; Sheaff et al., 2000; Jim et al., 2003; Ju et al., 2004; Forsthoefel et al., 2004; Li et al., 2006; Chen et al., 2007; . This implies the existence of alternative molecular signals (also named "degrons"), such as specific amino acidic sequence or structural elements, that mediate proteasome recognition and degradation of substrates independently on their ubiquitination levels (Baugh et al., 2009; .

The biological significance of ubiquitin-independent degradation of substrates by the 26S is a topic deserving great attention in order to decipher its physiological meaning in tissue homeostasis. Two proposed explanations envisage that it could be "only" a remnant of evolution, or else it could be rather a mechanism that provides, under selected circumstances, an alternative strategy to overcome Rpn4 and of proteasome in the case of inappropriate ubiquitin conjugation (Ju and Xie, 2004; Hanna et al., 2007; Erales and Coffino, 2014) .

In this regard, an intriguing example of how ubiquitin-dependent and ubiquitin-independent pathways cooperate to survey cellular homeostasis comes from the regulation of the proteome of lipid droplets (LDs), that are ubiquitous, endoplasmic reticulum-derived storage organelles from which neutral lipids are rapidly mobilized in response to cellular demands. In fact, some proteins of LDs are degraded by proteasome through the canonical ubiquitination pathway, whereas some others only when the "degron" signals they hold become unmasked upon protein insertion into the lipid monolayer (Bersuker and Olzmann 2017) . Interestingly, it has been reported that proteasome mediates ubiquitin-dependent degradation of patatin-like phospholipase domain-containing protein 3 (PNPLA3), whose sequence variant 148M is resistant to ubiquitination and to proteasome degradation, and accumulates into LDs, contributing to non-alcoholic fatty liver disease pathogenesis (Speliotes et al., 2011; Kozlitina et al., 2014; Basu Ray et al., 2019 ).

An additional issue in deciphering the mechanisms of proteasome degradation is the ubiquitinindependent degradation in vitro of macromolecular substrates by the uncapped 20S. In fact, several studies demonstrate that the 20S is able to degrade natively unfolded as well as oxidized and misfolded proteins (Davies, 1993; Pacifici et al., 1993; Grune et al., 1996; Reinheckel et al., 1998; Davies, 2001; Shringarpure et al., 2003; Raynes et al., 2016) . Indeed, oxidative stress induces chemical alterations, bringing about conformational changes and exposure of hydrophobic residues on damaged protein surfaces (Carrard et al., 2002; Raynes et al., 2016) . These surface hydrophobic patches stimulate, in an allosteric fashion, the translocation of the substrate into the 20S proteolytic chamber (see section 2.2 for details) (Giulivi et al., 1994; Coux et al., 1996; Davies, 2001; Kisselev et al., 2002) , since under oxidative stress conditions this form is more stable than the 26S, which is quickly and reversibly inactivated likely through dissociation into free 20S and 19S particles (Reinheckel et al., 1998; Reinheckel et al., 2000; Shringarpure et al., 2003; . Moreover, also the E1-E2-E3 cascade is transiently inactivated during oxidative stress, supporting a ubiquitin-independent degradation of oxidized proteins (Grune et al., 2011) .

Thus, the current view is that 20S activity on oxidized and damaged proteins might compensate for the loss of the ubiquitin-dependent activity of the 26S under redox imbalance.

intra-subunits interactions, constituting "the gate", which regulates the substrate access through a 13 Å entry pore into the antechamber (i.e, at α7-β7 interface). This passageway keeps the substrate in an unfolded state, directing it towards the catalytic chamber (i.e., at β7-β7 interface) Unno et al., 2002; Bajorek and Glickman, 2004; Marques et al., 2009; Ruschak et al., 2010;  Gaczynska and Osmulski, 2014) ( Figure 1C ). The insertion of the substrate through this "Nterminal gate" is the rate-limiting step of proteasome activity and prevents unwanted protein degradation (Akopian et al., 1997; Kohler et al., 2001) . In fact, RP binding induces the N-terminal tails displacement and opens the gate, facilitating the substrate translocation (see section 2.3) (Sledz et al., 2013; Matyskiela et al., 2013; Choi et al., 2016; Marshall and Vierstra, 2019) . However, it is worth recalling that RP binding to 20S is not an absolute requirement for proteasome activation, since 20S can switch from an inactive "closed" conformation to an active "open" conformation spontaneously or after chemical treatment (e.g., SDS) Forster et al., 2003; Bajorek and Glickman, 2004) . Noteworthy, since the α3 tail points toward the centre of the channel and maintains a close interaction with all other Ntermini of α subunits (Kohler et al., 2001a; Kohler et al., 2001b) , the deletion of first nine residues in α3 subunit N-tail in yeast 20S induces a general disorder in the neighbouring tails, stimulating the opening of the entry pore (Kohler et al., 2001a; Kohler et al., 2001b) . Thus, the α3Δn mutant is in a constitutively activated "open" state and its basal proteolytic activity toward small peptides is consistently enhanced, as compared to that of wild-type (wt) 20S (Kohler et al., 2001a; Kohler et al., 2001b; Bajorek and Glickman, 200) . Conversely, the double mutant α3-α7Δn more efficiently degrades macromolecular substrates with respect to either single mutants, suggesting that the interaction between these opposite tails is crucial in the regulation of gate opening (Bajorek et al, 2003; Bajorek and Glickman, 2004) . Interestingly, the α3Δn mutation does not alter the assembly of 26, as demonstrated by the evidence that the abundance and activity of mutant 26S are similar to those of wt-26S ; accordingly, human cell lines stably expressing α3ΔN subunits show enhanced activity of both free 20S and holoenzyme complexes.

This turns out in an increase of the degradation rate of poly-ubiquitinated proteins, reinforcing the Proteasome maturation refers to the process that drives the proper incorporation of individual subunits to assemble into a proteolytically active 26S. Whilst 19S assembly is largely uncovered yet (see section 2.3.2), the 20S assembly has been uncoiled at a great molecular detail also by virtue of the extensive similarity between yeast and mammalian pathways. This similarity has widened the repertoire of methodological approaches suitable to uncover the molecular insights. In eukaryotes, the stepwise recruitment of individual αand β-subunits to constitute a fully mature 20S requires the presence of five molecular chaperones, called Proteasome Assembly Chaperones (PAC1-4 in human, Pba1-4 in yeast) and Proteasome Maturation Protein (POMP in human, hUmp-1 in yeast) (Ramos et al., 2008; Le Tallec et al., 2007; Hirano et al., 2006) . These chaperones drive the sequential insertion of the subunits preventing the formation of off-target assemblies presumably through non-catalytic activities (Burri et al., PNAS 2000; Fricke et al., 2007, EMBO) . First, PAC1-PAC2 and PAC3-PAC4 work as heterodimers in recruiting the α-subunits during the earliest steps of biogenesis, that is the α-ring formation (Wu et al., 2018; Le Tallec et al., 2007; Hirano et al., 2006; Hirano et al., 2005; Matias et al., 2010) . Very recent advances in the field propose that in human cells α4, α5, α6 and α7 subunits first assemble to form a core tetrameric α-ring intermediate (α4-α7) , being driven by PAC3-PAC4 heterodimers, which localize at the inner side of the nascent α-ring (Wu et al., 2018; Satoh et al., 2019) . Recently, crystallographic data have allowed to identify a hydrophobic surface, surrounded by charged residues in PAC4, which is complementary to that of PAC3, thus providing a clue for the interaction between the two partners (Kurimoto et al., 2017) .

Notably, PACs surface was also found to display a charge complementarity with proteasomal α4 and α5 subunits, envisaging the first structural basis for the binding of the heterodimer PAC3-PAC4 to the nascent 20S (Kurimoto et al., 2017) .

Thereafter, the PAC1-PAC2 heterodimer binds the outer side of this assembly, favouring the recruitment of α1, α2 and α3 subunits, thus leading to the formation of a mature heptameric α-ring (Wu et al., 2018) . Besides correctly introducing the α-subunit, the presence of the chaperones prevents the formation of aberrant off-pathway α-ring dimers, an occurrence potentially favoured by is released and pro-β5, pro-β6 and pro-β1 subunits assemble (Hirano et al., 2006; Hirano et al., 2005; Satoh et al., 2019) . Remarkably, structural insights suggest that the pro-peptide is not merely involved in preventing the early activation of the catalytic Thr in the catalytically active subunit (see section 2.1), but is necessary for further stepwise incorporation of subunits, likely through an allosteric mechanism. The pro-peptides of β2 and β5 are essential for recruitment and incorporation of β3 and β6, respectively, whereas the β5 pro-peptide is necessary for the specific interaction with POMP (Hoefer et al., 2006) . The ultimate step of β-ring formation is the pro-β7 insertion and the formation of a half 20S (i.e., the 15S complex) which, upon dimerization, forms the mature 20S.

Although it is proven that full activation of 20S requires a) shedding of the β-subunits pro-peptides, b) PAC1-PAC2 detachment and/or clearance and c) POMP clearance, it is not fully clear whether the degradation of the chaperones is carried out by the 20S itself or if PAC chaperones are actually cleaved or released intact to be recycled for further maturation processes. It is further widely envisaged that additional unidentified factors may take part in the maturation process with activities overlapping with those of PACs depending on the cell metabolic needs.

The deepening of the molecular insights of proteasome maturation, both in terms of transcriptional regulation and of dynamics of proteins interaction, is expected to offer a new perspective for the development of therapeutic strategies based on the modulation of proteasome availability in selected tissues . Clinical and molecular studies envisage that increased POMP translation and bioavailability upon down-regulation of miR-101 (which targets POMP mRNA) is an oncogenic stimulus for breast cancer cells . Thus, the consequent increased proteasome intracellular content would confer protection from the proteotoxic insult to which highly proliferating cells are likely exposed, favouring cell survival .

Furthermore, POMP up-regulation enhanced the bulk proteasome activity under proteo-toxic conditions, providing a metabolic advantage under redox insult . As a matter of fact, recent genetic studies on POMP promoter have identified mutations at the 3'UTR region and splicing variants in different skin inflammatory disorders, such as CANDLE syndrome (Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated temperature) or proteasome-associated autoinflammatory syndrome (PRAAS). Furthermore, increased POMP levels were observed in psoriatic skin lesions (Dahlqvist et al., 2010; Ebstein et al., 2019; Poli et al., 2018;  ubiquitin (PRU) domain, whereas the C-terminal region of Rp4-Rpt5 extends out from the base body without interaction with other proteasome subunits, at least in the resting state ( Figure 2 ) (Djuranovic et al., 2009; Zhang et al., 2009; Husnjak et al., 2008; Tomko et al., 2010; Beck et al., 2012; Luan et al., 2016; VanderLinden et al., 2017; Budenholzer et al., 2017; Hemmis et al., 2019) . The first identified ubiquitin receptor was Rpn10, that is not considered part of the base, but functions as a bridge between the lid and the base, stabilizing their interaction (Martin et al., 2008; Maillard et al., 2011; Aubin-Tam et al., 2011; Beckwith et al., 2013; Erales et al., 2012) , as further suggested by the lid and base disassembly when Rpn10 is mutated (Deveraux et al., 1994; Isasa et al., 2010; Keren-kaplan et al., 2016) . Importantly, mono-ubiquitination of Rpn10, which is modulated by stressful conditions, regulates its association with proteasome, and thus proteasome activity and stability (Isasa et al., 2010; Keren-kaplan et al., 2016; Budenholzer et al., 2017) . An additional intrinsic ubiquitin receptor is the T1 toroidal region of the Rpn1 (Elsasser et al., 2004; Shi et al., 2016) , which, like Rpn10 and Rpn13, also recognizes ubiquitin like domains (UBLs) of extrinsic ubiquitin receptors (i.e., HR23/Rad23, PLIC2/DsK2 and Ddi1), stimulating the proteasome-mediated degradation of ubiquitinated substrates (Saeki et al., 2002; Leggett et al, 2002; Raasi et al., 2005; Shi et al., 2016; Spyracopoulos, 2016) . It remains unclear why proteasome contains such an array of ubiquitin-binding receptors, and what differential roles they might play in substrate recognition and degradation (Hamazaki et al., 2015; Bard et al., 2018; Cundiff et al., 2019) .

Upon recognition by intrinsic and extrinsic ubiquitin receptor, substrates are engaged with the AAA + motor of the highly dynamic Rpt1-6 hexameric ring that couples ATP hydrolysis to substrate unfolding and translocation, converting chemical energy into mechanical work (Pena et al., 2018; Eisele et al., 2018; Dong et al., 2019) . The C-terminal tails of Rpt2, Rpt3 and Rpt5 contain the conserved HbYX motif (see also section 2.3.2) that fits into the groove between adjacent α-subunits of 20S inducing a conformational change into their N-termini which drives 20S gate opening (Smith Panasenko and Collart, 2011; Park et al., 2011; De La Mota-Peynado et al., 2013; .

A main breakthrough for understanding the structural basis of 26S came from a series of Cryo-EM studies on proteasome holoenzyme from different species, such as yeast, rat and humans (Lasker et al., 2012; Lander et al., 2012; Matyskiela et al., 2013; Unverdorben et al., 2014; Wehmer et al., 2017; Wehmer and Sakata, 2016 \1q2 ; Bard et al., 2018) .

These studies have revealed the existence of at least four distinct human 26S conformational states (i.e., SA, SB, SC and SD, mirrored in yeast 26S by s1, s2, s3 and s4), that appear conserved among species. The numeric order of these main states is suggested by a structural comparison that reveals progressive and sequential movements from SA (s1) state through SB (s2) and SC (s3), to SD (s4), which is similar to SA ( Bard et al., 2018; Wehmer and Sakata, 2016) . In all identified conformations, the architecture and structure of 20S remains essentially unaltered, whereas the two subcomplexes of 19S, the lid and the base (see section 2.3.1) are highly dynamic, changing the relative orientation with respect to each other and to core particles; these movements are coupled to the functional cycle of 26S (Chen et al., Unverdorben et al., 2014; Wehmer et al., 2017) .

However, despite the advance in knowledge on 26S structure, we have to recall that an intriguing aspect, which has never been deeply investigated, concerns the conformational transition, occurring after the binding of the first 19S, on the opposite end of 20S, where one free α-ring surface is available for the binding of a second 19S particle, which yields a double capped 30S proteasome, whose real structure, as well as the function, remains poorly understood .

The yeast s1 is a low energy ATP-bound ground state, that is assumed to be the primary substratebinding conformation (Matyskiela et al., 2013; Sledz et al., 2013; Unverboden et al., 2014; Wehmer et al., 2017; Ding et al., 2017; Zhu et al., 2017) . In the s1 state, the 20S gate is closed, since the substrate translocation channel of ATPase ring is not aligned with the 20S gate, and the active site of Rpn11 is 25Ǻ away from substrate entry pore (Wehmer et al., 2017; Eisele et al., 2018; Finley and Prado, 2019) . The transition toward the s2 state is driven mainly by the lid rotation, which drives Rpn11 to a position above the central processing pore of the base. On the other hand, the progression from s2 to s3 is mediated by a rearrangement of Rpt1-Rpt6, wherefore N-ring of Rpts and AAA+ domains shift toward Rpn1, thus generating a wider channel aligned with core particle axial pore (Matyskiela et al., 2013; Unverboden et al., 2014; Wehmer et al., 2017; . Therefore, the s3 state is characterized by the axial alignment of the essential J o u r n a l P r e -p r o o f DUB Rpn11 (see also section 2.3.1), 19S translocation channel, and 20S gate. However, in spite of these rearrangements and of the evidence that s2 and s3 states are primed for substrate degradation, the 20S gate is still mostly occluded, preventing substrate entry (Matyskiela et al., 2013; Sledz et al., 2013; Bard et al., 2018; Finely and Prado 2019) . The gate becomes fully opened only during the transition from s3 to s4, inducing the entry of the substrate into the catalytic core; thus, gate opening is a consequence of the insertion of "HbYX" motif of C-termini of Rpt2-Rpt3-Rpt5 subunits into 20S pocket (see section 2.3.1). Stable docking of HbYX motifs into the 20S is insufficient to promote gate opening, which is completed only in the s4 state upon engagement of the C-termini of Rpt6 and Rpt1 into the α-ring (Eisele et al., 2018; Finley and Prado, 2019) . Besides these four states, further structural and biochemical studies have revealed recently the presence of two additional open gate states in yeast proteasome (i.e., s5 and s6) (Eisele et al., 2018) . In the case of human 26S proteasome Cryo-EM studies showed that the substrate Sic1 PY (i.e., the Cdk inhibitor Sic1 from Saccharomyces cerevisiae with a Pro-Pro-Pro-Ser motif inserted into N-terminal) exists in seven conformational states, EA1, EA2, EB, EC1, EC2, ED1, ED2 (Dong et al., 2019) . EA1 and EA2 states represent two initial ubiquitin recognition states; EB2 is the "de-ubiquitination" state, in which the iso-peptide bond between Rpn11 and substrate is close to the zinc-active site of Rpn11; EC1 and EC2 are conformations at the onset of substrate translocation; ED1 and ED2 carry on and complete substrate translocation (Dong et al., 2019) . Functional models of 26 activity, derived both from cryo-EM and biochemical analysis, couple ATP hydrolytic cycle to substrate translocation (Matyskiela et al., 2013; de la Pena et al., 2018; Dong et al., 2019) . Therefore, sequential ATP hydrolysis and phosphate release, which are coordinated within the ATPase motor, seem to supply "the power" to induce conformational changes that drive the substrate through the central pore (de la Pena et al., 2018; Eisele et al., 2018; Tundo et al., 2018; Dong et al., 2019) . In agreement with a "rotatory" mechanism, a hydrolytic event in a single Rpt subunit is followed by another one in the nearby subunit, thus proceeding throughout the entire ring (de la Pena et al., 2018; Eisele et al., 2018; Tundo et al., 2018; Dong et al., 2019) . In fact, it has been proposed that a specific Rpt subunit binds ATP and engages substrate at the uppermost position; then, this subunit hydrolyses ATP (when at the penultimate position of the staircase), releasing the phosphate moiety and disengaging from substrate, which proceeds to the next hydrolytic step (Eisele et al., 2018; de la Pena et al., Importantly, cryo-electron tomography approaches have also visualized proteasome particles in their native conformation in living cells, allowing to have an insight on the percentage of different populations that harbour the cells (Asano et al., 2015; Guo et al., 2018; Finley and Prado, 2019) . In intact hippocampal neurons, a molecular census of proteasome conformational states showed that, in the absence of proteotoxic stress, only 20% of the 26S was engaged in substrate processing, whereas the remaining portion was in the substrate-accepting ground state; it suggests that the capacity of the proteasome system is not fully exploited by the cell under physiological conditions (Asano et al., 2015) . Interestingly, poly-Gly-Ala (poly-GA) aggregates, which result from aberrant expansion of GGGGCC repeat in C9orf72 gene (i.e., the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia), recruits 26S molecules which are in the s4 state (Guo et al., 2018) , unlike the general pool of proteasome. However, since poly-GA are not favourable proteasome substrates, 26S sequestration and consequent inhibition has been proposed to cover a crucial role in neurodegeneration (Guo et al., 2018; Finley and Prado, 2019) .

Over the last decades, hallmarks of cancer cells have been described to provide a sort of universal definition which would account for the multi-step development of human tumours (Hanahan and Weinberg, 2011) . These hallmarks, which are complementary features that enable tumour growth and metastatic dissemination, include proliferative signalling, growth suppressors inactivation, cell death resistance, replicative immortality, angiogenesis, invasiveness and dissemination, cell metabolism reprogramming and immune-surveillance evasion (Hanahan and Weiberg, 2000; Hanahan and Weinberg, 2011; Tundo et al., 2019) . Recently, resistance to proteostasis unbalance has been proposed as a new malignant hallmark of cancer, envisaging the possibility that this acquired property cooperates with the other altered circuits to allow cancer cell survival, progression, novel properties to promote their survival (Calderwood et al., 2006; Vahid et al., 2017) .

In recent years, three main reasons have gained considerable insight as to why PNs are altered in human tumours, namely 1) genomic instability; 2) persistence of stressful conditions in the tumour micro-environment, and 3) age-related proteome imbalance (Dong and Cui, 2018) .

First, cancer cell genome is highly unstable and builds up several point mutations in protein coding sequence and/or genome mutations (e.g., large duplications, deletions, inversions, and translocations as well as altered copy numbers of entire chromosomes, such as aneuploidy). This may turn out in an inappropriate repression or activation of tumour suppressors and oncogenes, respectively, excessive protein synthesis, and/or translation of mutated proteins with altered folding, function and turn-over (Weaver and Cleveland, 2006; Benbrook and Long, 2012; Vogelstein et al., 2013; Kim and Zaret, 2015) . It has been estimated that over 90% of human solid tumours harbour aneuploidies that lead to an excess in protein synthesis (Weaver and Cleveland, 2006; Williams and Amon, 2006; Dai et al., 2012) ; indeed, this is a relevant issue mainly for proteins that become functional upon assembly in stoichiometric complexes such as in the case of ribosomes (Deshaies et al., 2014) . Therefore, genomic alterations support a proteostasis unbalance (also referred as proteotoxic crisis) that renders cancer cells more dependent than normal cells on PNs clearance mechanisms, including UPS (Deshaies et al., 2014) . Accordingly, yeast cells with one-third of single chromosomal aneuploidies are hypersensitive to proteasome inhibitors, and some cells "adapted" to aneuploidy harbour mutations that depress UPS activity (Torres et al., 2007; Torres et al., 2008; Torres et al., 2010) .

Secondly, during tumour development, tumour cells are continuously exposed to a variety of extrinsic perturbations, such as nutrient deprivation, hypoxia, and acidosis. Despite this pressure, tumour cells successfully proliferate and efficiently withstand this challenge by adapting to the fluctuations of the microenvironment, reprogramming their proteome and fully exploiting the cell defence mechanisms against proteotoxic stress. Thus, ultimately, stressful conditions lead to a disruption of the proteostasis balance, which is associated to the promotion of malignant properties (such as invasiveness, immune surveillance escape, and metabolism reprogramming), achieving a plethora of PN alterations (Oromendia and Amon 2014; Dufey et al., 2015; Nam and Joe, 2019) . culminates in PNs alteration (Sklirou et al., 2018; Dong and Cui, 2018) . Thus, in a vicious circle, unbalanced PNs lead to the proteotoxic crisis, which favours tumourigenesis (Miller et al., 2014; Arnsburg et al., 2014) . As a matter of fact, in accordance with the proteotoxic crisis hypothesis, reprogramming the proteome might represent a novel therapeutic approach, since agents that target components of different PN pathways are expected to be more toxic for cancer cells than for normal cells (Deshaies, 2014; Yuan et al., 2018) . In the next paragraphs, we will review the biological rationale for targeting proteasome in the context of UPS as a strategy to treat cancer.

A number of preclinical studies have reported alterations of proteasome expression and activity in different type of cancers, including haematological malignancies, lung, breast, pancreatic, head and neck, and thyroid cancers (Kumatori et al., 1990; , Chen et al., 2005 Artl et al., 2009; Roeten et al., 2018) . The reason of this high proteasome activity is not well understood, even though it is likely linked to stressful conditions (e.g., hypoxia, reperfusion, alteration of growth factors and cytokines levels), which evolve in the context of a heterogeneous tumour microenvironment. Deregulation of the proteasome activity can destabilize and/or disrupt the balance between tumour suppressors and oncoproteins, promoting cancer progression (Ogiso, 1999; Kaplan et al., 2017, Chang and Ding, 2018) . An element of complexity in understanding the role of proteasome in carcinogenesis is also represented by the fact that most investigations are carried out in unsorted cancer cells, which do not include cancer stem cells (Voutsadakis, 2017) ; thus, cancer stem cell theory states that all tumour cells derive by a small percentage of cancer stem cells capable of repopulating tumours after therapy (Simons and Clevers, 2011; Hanahan and Weinberg, 2011) . Noteworthy, the proteasome function is decreased in these cells with respect to the bulk of tumour population, revealing that a better understanding of proteasome regulation in different cell sub-sets might unveil further opportunities in cancer therapy (Banno et al., 2016; Voutsadakis, 2017) . Despite the criticism, there are many key proteins, degraded by proteasome, that are involved in carcinogenesis; below are listed examples of proteins, which are considered crucial in cancer progression and are reported to mediate cell death after exposure to proteasome inhibitors (Ciechanover et al., 2001; Evan and Vousden, 2001; Soave et al., 2017; Johnson et al., 2015; Jang et al., 2018) (Figure 3 ).

J o u r n a l P r e -p r o o f NF-kB is a crucial transcription factor that induces the expression of a wide range of genes involved in cell proliferation, apoptosis, inflammation and angiogenesis (Karin et al., 2002; Wu and Shi, 2013; Qureshi et al., 2018) . Alteration of NF-kB pathway has been documented in a series of human tumours, including breast, lung, prostate, pancreatic cancer and melanoma, as well as in haematological malignancies, such as Hodgkin's/Non-Hodgkin's lymphoma and multiple myeloma (Kim et al., 2006; Karin et al., 2005; Braun et al., 2006; Aggarwal et al., 2004; Van Waes 2007; Johnson 2015; Perkins 2012; Kaplan et al., 2018) . It is generally accepted that NF-kB promotes cancer progression by inhibiting apoptosis, and that also chemo-and radiotherapy treatments activate NF-kB signalling, inducing acquired resistance to conventional cancer therapy (Baldwin, 2001; Nakanishi and Toi, 2005; Wu and Shi, 2013) . Under unstimulated conditions, NF-kB homoor hetero-dimers are sequestered in an inactive form in the cytoplasm by its inhibitor IkB. Different stimuli, including stress and chemotherapy, activate IkB kinase (i.e., IKKB) that phosphorylates IkB, leading to its ubiquitination and degradation by the proteasome. Free NF-kB dimers then translocate into the nucleus wherein they induce the transcription of target genes (Baldwin et al., 1996; Schwartz et al., 1999; Traenckner et al., 1995) (Figure 3 ). Mammals express five NF-κB proteins, namely RelA (p65), RelB, c-Rel, p50 and p52. Proteasome is involved in the maturation process of p50 and p52, which are synthesized as large precursors of p105 and p100 respectively (Fan et al., 1991; Beinke and Ley, 2004) . Treatment with proteasome inhibitors (PI), such as bortezomib, blocks p105 and p100 processing, and/or IkB degradation, thus inhibiting the NF-kBmediated cancer promoting activity (Kaplan et al., 2017; Wu8 et al, 2013; Johnson et al., 2015) . Indeed, NF-kB activation seems to play a major role in the antitumor effect of bortezomib, particularly in multiple myeloma and melanoma cells (Hideshima et al., 2002; Amiri et al., 2004) . Among the numerous proteins regulated by NF-kB signalling, cyclin D1 plays a crucial role in cancer progression, since it is a key regulator of late G1 phase of cell cycle. The cyclin D1-Cdk4/6 complexes generate the phosphorylated form of the Rb protein, resulting in the release of EF2 transcription factors, inducing its activation. This is followed by the expression of cyclin E, which interacts with Cdk2 bringing about the hyper-phosphorylation of Rb, cyclin A and genes involved in DNA synthesis. These steps anticipate the S phase progression.

P53 is a nuclear transcription factor that regulates apoptosis, DNA repair, angiogenesis, cell growth and senescence (Vogelstein et al., 2000; ; thus, regulation of its level is fundamental to guarantee cell homeostasis. This protein is characterized by a very rich functional spectrum that is the consequence of a structural complexity which renders it able to interact with a myriad of partners. P53 exists as a dynamic ensemble of different "proteoforms", and this structural plasticity is due to the presence of intrinsically disordered regions, as well as to several modifications at transcriptional and post-translational level. Several p53 mutants form amyloid structures that aggregate in the cell in a "prion-like" fashion with a gain-of-function effect (Rangel et al., 2019) . It is noteworthy that p53 unfolded mutant forms are shared in cancer and in Alzheimer's disease (AD) tissues, actually entering in the list of biomarkers that can be used for their diagnosis (Amor-Gutierrez et al., 2020) Under normal conditions, p53 degradation is a complex and finely regulated process, which is predominately orchestrated by the MDM2 protein, a RING-finger E3-ligase that promotes the polyubiquitination of p53, and, thus, its degradation by the 26S (Figure 3 ) (Haupt et al., 1997; Momand et al., 2000; Poyurovsky et al., 2007; Brown et al., 2009; Devine et al, 2013) . P53 pro/apoptotic function covers a prominent role in tumour suppression, and mutations of p53 gene are among the most frequent genetic events in human tumours (Kandoth et al., 2013; Walerych et al., 2015; Walerych et al., 2016; Niazi et al., 2018) . Additionally, tumours expressing wt-p53 often have different mechanisms to bypass its activity, such as the overexpression of MDM2 Chene et al., 2003; Quesnel et al., 1994) . A series of studies, performed in different cancer cell models, including melanoma, head and neck and colon cancer, reveal that one of the main mechanisms of cell death induction by proteasome inhibition, is the p53 pathway stabilization (Fernandez et al., 2005; Qin et al., 2005b; Zhu et al., 2005; Concannon et al., 2007; Gomez-Bougie et al., 2007; Voortman et al., 2007; Li et al., 2008; Morsi et al., 2018; Lopes et al., 1997; MacLaren et al., 2001; Yu and al., 2007) . Accordingly, pro-apoptotic factors, such as Noxa and Bax, are primary p53-responsive elements ( Figure 3 ) (Oda et al., 2000; Albert et al., 2014) . However, controversial results are still reported, since the killing of some cancer cells was shown to involve a p53-independent mechanism of Noxa induction, providing evidences for a novel strategy to bypass the apoptotic resistance of tumour cells (Perez-Galán, 2006; Qin et al., 2005; Strauss et al., 2007; Devine and Dai, 2013; Yerlikaya et al., 2012; 

J o u r n a l P r e -p r o o f One of the main hallmarks of carcinogenesis is the loss of cell division control. Proteasome is involved in the regulation of the cell cycle, since it degrades cyclin dependent kinases (Cdk) and

Cdk inhibitors (CdkIs) (Glickman and Ciechanover, 2002; Diehl and Ponugoti, 2010) . Generally, the main function of CdkIs consists in the inhibition of cyclin/Cdk complexes, blocking cell division; p21 and p27 CdkIs expression is frequently suppressed in cancer, favouring the dysregulation of cell proliferation (Chu et al., 2008, Abbas and Dutta, 2009 ). P27 is a well-known negative regulator of cell cycle progression in mammalian cells which binds and suppresses the activity of two crucial complexes (i.e., Cdk2/cyclin E and Cdk2/cyclin A), mediating G1 progression and G1/S transition (Sherr and Roberts; Slingerland and Pagano, 2000) . P27 is ubiquitinated by the E3-ligase complex SCF Skp2 and then degraded by the 26S (Abbas and Dutta, 2009; Chu et al., 2008; Slingerland and Pagano, 2000; Rastogi and Mishra, 2012) . Low levels of p27 are reported in different cancers (including prostate, breast, and colorectal), as a consequence of an increased UPS activity, which leads to its accelerated degradation (Loda et al., 1997 , Tsihlias et al., 1999 Glickman and Ciechanover, 2002) . Moreover, consistent with the oncogenic role of SCF Skp2 , its overexpression is associated with low levels of p27, and thus with the deregulation of cell cycle progression (Gstaiger et al., 2001; Lee et al., 2016b) . P21, whose stability is essential for cell fate decision, binds Cdk2/cyclin E complex (blocking the onset of the cell S phase) and cyclin B/Cdk1 complex (leading to a growth arrest in the G2 phase) (Abbas and Dutta, 2009; Rastogi and Mishra 2012) ( Figure 3 ). Moreover, p21 binds the proliferating cell nuclear antigen (PCNA), interfering with PCNA-dependent DNA polymerase activity, inhibiting DNA replication and modulating PCNA-dependent DNA repair processes (Mortusewicz et al., 2005; Walsh and Xu, 2006; Moldovan et al., 2007; Abbas and Dutta, 2009 ).

Under normal conditions, p21 levels are controlled at a transcriptional level mainly by p53. In several cancer types, proteasome inhibition brings about accumulation of p53, enhancing its nuclear export, and thereby the expression of transcriptional target genes, including p21, counteracting the proliferation stimulus associated to low p21 levels (Brugarolas et al., 1995; Deng et al., 1995; Roninson, 2002; Eastman, 2004; Abbas and Dutta, 2009) . Beside a transcriptional control by p53, p21 levels are modulated through either (i) a ubiquitin-dependent degradation by the 26S, and (ii) a ubiquitin independent pathway of degradation by the uncapped 20S, which has been proposed for the free form of p21 ( Figure 3 ) (Sheaff et al., 2000; Touitou et al., 2001; Li et al., 2007; Chen et al., 2007; Deng et al., 2018) . In particular, case (i) requires p21 ubiquitination by three E3 ligases (i.e., SCFS KP2 , L4 CDT 2 and APC/ C CDC20 ) at specific stages in an unperturbed cell cycle, which occurs only when p21 is bound to cyclin/Cdk complexes, and PCNA. Consistent with these studies, J o u r n a l P r e -p r o o f proteasome inhibition has been reported to considerably increase the intracellular level of p27 and p21 in many cancers, favouring cell cycle arrest (Sterz et al., 2010; Mi et al. 2011; Huang et al., 2011; Rastogi and Mishra 2012; Li et al., 2018) .

Although more than one thousand proteins belong to the UPS function in the ubiquitination and recognition of ubiquitinated protein substrates, the vast majority of currently available inhibitors, which have been designed and synthesized to block this pathway, target the proteolytic core of 20S.

These proteasome inhibitors are broadly categorized into different groups, according to the origin (e.g., synthetic or natural products), the kinetic mechanism of inhibition (e.g., competitive or noncompetitive) or else the chemical structure/reactivity. This chapter and the following one will deal with the discussion on the most promising and clinically available inhibitors, pointing out, wherever possible, their molecular action as well as their pharmacological profile and therapeutic outcome of their usage in clinic.

Proteasome inhibitors were initially developed to prevent cancer-related cachexia, in view of proteasome role in protein turnover (Manasanch and Orlowski, 2017) . To date, UPS is universally considered a "bona fide" target for the development of anti-cancer drugs (Adams, 2004a,b; Cloos et al., 2017; Gandolfi et al., 2017; King et al., 1996; Landis-Piwowar et al., 2006; Niewerth et al., 2015; Bullova et al., 2017; Roeten et al., 2018; . Indeed, PIs represent the reference treatment of multiple myeloma (MM), in view of its high sensitivity to this class of anticancer agents (Chauhan et al., 2005; Roccaro et al., 2006; Fricker, 2020; Gandolfi et al., 2017; . It is important to recall that MM is an aggressive and often incurable plasma cell dyscrasia characterized by uncontrolled proliferation of abnormal plasma cells, which invade the bone marrow, producing abnormal monoclonal immunoglobulins, which circulate in blood. The poor prognosis of MM, which reflects the genomic complexity of the disease, has dramatically improved after the introduction of PIs in disease management, mainly for patients displaying a refractory MM (RMM) and relapsed and refractory MM (RRMM), as discussed in the next section Leleu et al., 2018) . As reported previously (see Section 3.2), proteasome inhibition results in multiple deleterious downstream effects in cancer cells, including down-regulation of NF-κB signaling, stabilization of p53, cell cycle arrest, which all lead to apoptosis. Moreover, PIs downregulate adhesion molecules and secretion of cytokines (Read et al., 1995) , inhibit angiogenesis (Sunwoo et al., 2001) and induce DNA-damage (Łuczkowska et al., 2020) .

The effort to develop PIs has a long history and many different approaches have been adopted, ranging from the use of endogenous and/or natural compounds to the synthesis of new ones (Buac et al., 2013) . Initially, proteasome targeting for cancer therapy has been viewed with scepticism, mainly because of the fundamental and crucial roles of proteasome in regulating cell homeostasis in all living cells (Park et al., 2018) . Although the reason for the increased cytotoxicity of PIs on proliferating tumour cells is not completely understood, it is widely reported (Chauhan et al., 2005) that cancer cells are more dependent on proteasomal activity, likely because of the higher protein turnover they experience, thus being also more sensitive to its blockage (Almond and Cohen, 2002) .

Tumour cells have a proteasome pathway more active than normal cells, since an increased capability for synthesis, movement and modification of proteins is necessary to preserve their uncontrolled cell proliferation and their high metastatic potential high capacity (Chen and Madura, 2005 Fricker, 2020; Gandolfi et al., 2017) . Although the availability of PIs has led to an improvement of patients' survival rate, the therapeutic potentiality of these drugs is limited by several drawbacks, including the low potency and specificity of approved molecules, adverse effects and development of drug resistance (Assaraf et al., 2019; Cree and Charlton, 2017; Gacche and Assaraf, 2018; Gonen and Assaraf, 2012; Wijdeven et al., 2016; Zhitomirsky and Assaraf, 2016) . Furthermore, the therapeutic potential of bortezomib is negatively affected by pharmacokinetic issues and by the very limited distribution to solid tumours which require exceedingly high and toxic doses (Huang et al., 2014; Grigoreva et al., 2015) . The use of more recently approved carfilzomib and ixazomib has only partially allowed to overcome these issues (see Section 3.3.2.1). Therefore, there is a growing demand of novel inhibitors with different mechanisms of action and more favourable pharmacological profiles. Additionally, it emerges that the antitumor activity of PIs is markedly improved in combination with conventional therapeutic strategies or with other molecular targeting agents, such as cell surface death receptor, autophagy, STAT3 and HDAC inhibitors (Pei et al., 2004; Li et al., 2008; Li et al., 2009; Li et al., 2010a; Seki et al., 2010; Yoshiba et al., 2011; Li and Johnson, 2012) . Accordingly, a number of preclinical and clinical studies are ongoing to evaluate further new drug combinations, and to optimize administration schedules of therapeutic protocol J o u r n a l P r e -p r o o f already used (Berenson et al., 2007; Chen et al., 2011a; Johnson, 2015; Wallington-Beddoe et al., 2018) .

Generally, PIs are electrophilic molecular species that react covalently with the threonine residues of the proteasome active sites (Harer et al., 2012) . The first PIs were analogs of serine protease inhibitors, characterized by hydrophobic peptide aldehydes, mimicking substrates of the proteasome β5 active site and reacting with the nucleophilic hydroxyl group of threonine to form reversible hemiacetal adducts. However, first aldehyde inhibitors turned out to have additional targets in the cell besides the proteasome, also inhibiting cathepsin B and calpains (Kisselev & Goldberg, 2001) .

For this reason, other molecular scaffolds have been investigated and peptide boronates (such as bortezomib and ixazomib) as well as epoxyketones (i.e., carfilzomib and oprozomib) have been synthesized. These new and more specific PIs have experienced great success in clinics (as extensively discussed in section 3.3.2), and most of them are currently used as therapeutic drugs, even though some of them still retain activity towards non-proteasome targets. In fact, bortezomib and most second-generation boronates also co-inhibit caspase-like sites (Kisselev et al., 2012) . In the next section, the main chemical properties of PIs approved and/or ongoing in clinical trials are discussed.

Bortezomib is a dipeptide containing phenylalanine and leucine with a boronic acid instead of a carboxylic acid, and a pyrazinoic acid moiety to protect the N-terminus. The structure of bortezomib bound to the 20S has been solved, elucidating the binding mode and mechanism of action at the molecular level (Groll et al., 2006) (Figure 4A ). Bortezomib binds reversibly to the chymotryptic-like (CT-L) β5 subunit of the proteasome, even though it has also been reported to bind the caspase-like (C-L) β1 and trypsin-like (T-L) β2 subunits with lower affinity (Buac et al., 2013) ; however, a good selectivity of bortezomib towards specific proteasome subunits is dictated by the composition of their substrate binding pockets, which differs in the three catalytic β-subunits.

In the presence of bortezomib, an anti-parallel β sheet conformation is adopted by domains in the catalytic clefts, and direct hydrogen bonds are formed between the conserved residues (i.e., Gly47N, Thr21N, Thr21O, and Ala49O) of the proteasome β-type subunits and the main chain atoms of the drug, stabilizing the complex ( Figure 4A ). The boronic acid is responsible for the J o u r n a l P r e -p r o o f actual inhibition, ensuring an increased specificity for the proteasome. Indeed, the boron atom covalently binds the oxygen of Thr1Oγ (the electrophilic functional group that normally reacts with peptide bonds of substrates, see section 2.2.1), while the acidic boronate hydroxyl groups are bound to Gly47N, bringing about a stabilization of the oxyanion hole. Further stabilization of the tetrahedral boronate adduct comes from a second acidic boronate hydroxyl moiety, which works as a catalytic proton acceptor and is H-bridged to the N-terminal threonine amine atom. A wide range of specific inhibitors has been developed, but usually peptide boron esters and acids are powerful inhibitors of serine proteases, as they interact covalently but reversibly with the active hydroxyl site of this class of the enzymes (Harer et al., 2012) . Furthermore, these peptide boron esters are less reactive toward circulating nucleophiles in aqueous solutions than their aldehyde counterparts (Adams et al., 1998) .

Bortezomib induces toxicity in cancer cells through different mechanisms, including (i) inhibition of the NF-kB pathway, which has been envisaged as the main target of bortezomib clinically efficacy; (ii) stabilization of p53 pathway, which leads to apoptosis mainly by increasing the level of proapoptotic factors, such as NOXA and Bcl-2; (iii) modulation of CdkIs levels. Moreover, bortezomib inhibits tumor angiogenesis probably as a result of reduced vascular endothelial growth factor receptor (VEGFRs), which seems to be linked to the inhibition of NF-kB (Hideshima et al., 2001; Sunwoo et al., 2001; Qin et al., 2005; Pandit and Gartel, 2011; Nunes & Annunziata, 2017) . Despite the plethora of mechanisms of actions responsible for the high toxicity of bortezomib towards cancer cells and the high specificity towards serine proteases, peptide boron esters containing acids, such as bortezomib, can become bioactivated to chemically reactive imine amide metabolites inducing drug toxicity (A. C. . As a matter of fact, carbinolamides metabolites have been detected after incubation with human liver proteins and the formation of GSH conjugates was also observed, both likely stemming from electrophilic reactions of the imine amides with the nucleophilic GSH. The observed metabolites seem to be produced via oxidative de-boronation, catalyzed by hepatic cytochrome P450 enzyme, and bortezomib toxicity has been ascribed to their formation and high reactivity (see also section 3.3.2.1). A way to reduce these adverse effects of bortezomib treatment is the use of appropriate methods for administering this agent, such as early-dose reduction and once-weekly and subcutaneous administration.

A crucial drawback, encountered when bortezomib is used as a therapeutic drug, is the rapid development of resistance in response to the treatment (Barrio et al., 2018) . Many studies have described a plethora of strategies the cancer cells may evolve to acquire bortezomib resistance. In J o u r n a l P r e -p r o o f this regards, selective down-regulation of specific 19S subunits and the consequent reduced flux of substrates through proteasome has been reported to be a major strategy several cancer cells may adopt to cope with proteasome inhibition (Tsvetkov et al., 2015; Tsvetkov et al., 2017) . Somatic mutations in the catalytic cleft of β5 and involved in the binding to bortezomib have been further described in patients with MM who underwent prolonged therapy with PIs: in this case, resistance induction was acquired through missense mutations and resistance was effective, though at a variable extent, also to next generation PIs (see next paragrapshs) (Barrio et al., 2019) .

Further, a selective overexpression (up to 60-fold) of a mutant β5 protein has been proposed at the origin of the bortezomib resistance, whereas marked changes in CT-L proteasome activity are not found (Oerlemans et al., 2008) . On the other hand, other studies have reported a significant decrease of the CT-L proteasome activity after 1 h in four different cell lines, maintaining such an inhibitory activity for as long as 24 h (Bonvini et al., 2007) . Furthermore, an increase in the accumulation of the β5 precursor form was observed, even though no significant alteration in the expression profile of the mature form was detected (Yerlikaya & Okur, 2019) . Analogously, it has been also reported that α5 promotes the tumourigenic process of prostate cancer cells and is linked to bortezomib resistance (Fu et al., 2019) . Other changes in bortezomib resistant cell lines, such as increased expression of β1 and β5 proteasome subunits, upregulation of pro-apoptotic proteins of the Bcl-2 protein family, Bax and Noxa have been also reported . Moreover, the lack of some proteins, such as XBP1, which decreases the endoplasmic reticulum burden and affects the unfolded protein response, has also been proposed as a possible cause of bortezomib resistance (Fall et al., 2014) . Interestingly, certain factors have been proposed as predictive markers of response to bortezomib treatment. Among others, KLF9, CDK5, Nampt and accumulation of unfolded proteins in the endoplasmic reticulum (ER stress) and UPR-associated markers (XBP1, ATF3, and AFT4) have been identified to play an important role in bortezomib sensitivity. It is therefore clear that further studies are demanded in order to better understand the underlying mechanisms which limit the use of this compound for cancer treatment.

In general, epoxyketone PI are characterized by a short peptide core, and a terminal α,βepoxyketone dual electrophilic reactive warhead, which determines their activity (Schrader et al., 2016) . The most important representative of this PI class is carfilzomib, a tetrapeptide with a terminal epoxyketone group, which seems to be highly specific for the proteasome (see sections 3.3.1.1 and 3.3.2.2.2) (Muz et al., 2016) , It displays an inhibitory power equivalent to that of bortezomib for CT-L subunits of the proteasome (IC 50 = 6 nM), whereas C-L and T-L sites are only very weakly inhibited by carfilzomib (IC 50 = 2400 and 3600 nM, respectively); thus it is considered a selective inhibitor of CT-L activity (Demo et al., 2007; Boccon-Gibod et al., 2020) . Carfilzomib forms a covalent adduct between its C-terminal ketone moiety and Thr1O of each inhibited subunit ( Figure 4B ). Additionally, unlike peptide boronates, such as bortezomib and ixazomib, carfilzomib forms a stable morpholine ring between Thr1 N-terminal amino group and epoxide α carbon. These further covalent interactions dramatically enhance the specificity of epoxyketones for proteasome with respect to other proteases, making this PI class essentially irreversible under physiologicaltreatment conditions (Huber et al., 2012; . Crystallographic studies on human 20S proteasome in complex with carfilzomib clarify the structural basis for the high in vivo drug's selectivity for CT-L activity. The high specificity for CT-L activity can be attributed to van der Waals interactions between carfilzomib and S1, S3, and S4 pockets of β5 subunits, whereas in the T-L β2 subunit, carfilzomib forms favorable van der Waals interactions only with S3 and S4

pockets, but not with S1 ( Figure 4B ). In fact, the main differences between CT-L and T-L sites mostly reside in the S1 pocket size, which is much wider in the T-L subunit than in the CT-L one, rendering less effective the van der Waals contacts between the P1 leucyl group of carfilzomib and the S1 pocket of the T-L subunit. In addition, His116 of β7 subunit sterically blocks the entry of carfilzomib P4 phenyl group into the S4 pocket of the T-L subunit, shifting P4 up to 3.7 Ǻ away from the S4. Furthermore, the polarity of the C-L S1 pocket impairs an interaction with the carfilzomib hydrophobic P1 leucyl group ( Figure 4B ). As a whole, these differences lead to a disordered N-terminus of carfilzomib, likely contributing to the higher IC 50 value of C-L activity (Harshbarger et al., 2015) .

The mechanisms through which carfilzomib induce cell death are less known than for bortezomib, even though several studies demonstrated that it elicits programmed cell death acting in different ways, such as (i) activating c-Jun-N-terminal kinase, (ii) promoting mitochondrial membrane depolarization and favouring cytochrome c release, (iii) increasing the levels of pro-apoptotic factor Noxa, and activating caspase-3 and caspase-7 (Parlati et al., 2009 , Naraynan et al., 2020 . The introduction of carfilzomib in clinical therapy has allowed to overcome some criticisms related to bortezomib administration, like a reduced incidence of adverse effects (e.g., severe peripheral J o u r n a l P r e -p r o o f neuropathy) and acquired resistance; therefore, it has become a key option for the treatment of refractory MM patients (see section 3.3.2.2.1). However, a number of patients also display intrinsic resistance or develop resistance to carfilzomib treatment (Shah et al., 2018) . The reasons for this might stem from mutations or overexpression of proteasome catalytic subunits, but a likely contributor to carfilzomib resistance could also be the overexpression of the efflux pump Pglycoprotein (P-gp), reducing the drug intracellular concentration, since carfilzomib is a recognized substrate of this enzyme (Ao et al., 2012; Zang et al., 2014; Besse et al., 2019; Zheng et al., 2017;  Lee er t al., 2019).

Oprozomib is an orally bioavailable peptide epoxyketone, currently tested in ongoing clinical trials.

It is a tripeptide structural analogue of carfilzomib, which was synthesized to improve drug absorption. In fact, it is thought that smaller peptides are absorbed more effectively across the small intestine wall (Hamman et al., 2005; Zhou et al., 2009) . Like carfilzomib, oprozomib primarily exhibits irreversible binding kinetics to CT-L subunit (Rajan and Kumar, 2016) , and the co-crystal structure of human 20S and oprozomib enhanced the knowledge of how proteasome active sites interact with peptide epoxyketone inhibitors (Schrader et al., 2016) . Oprozomib, like other epoxyketone inhibitors, forms a seven-membered, 1,4-oxazepano adduct with the catalytic Thr within the β5 active site, whereas (as also reported for carfilzomib) previous findings reported the formation of a 1,4-morpholino adduct. Therefore, these novel solved structures have indicated that, during the second step of the inhibitory reaction, the Thr N-terminal amino group attacks the β carbon rather than the α carbon of the inhibitor's epoxide (Schrader et al., 2016; Carmony et al., 2017) . Concerning the mechanism through which oprozomib mediates cancer cell death, it has been demonstrated that oprozomib induces apoptosis through the activation of caspase 3, 8 and 9 (Chauhan et al., 2015) , PARP cleavage, and, interestingly, it seems to block angiogenesis that it is known to play a key role in MM progression (Podar et al., 2001; Chauhan et al., 2010; Giuliani et al., 2011; Zhu et al., 2019) .

Ixazomib and delazomib are both orally available structural analogues of bortezomib with a boronic acid as pharmacofore. In particular, ixazomib, which is currently approved by EMA and FDA, is a dipeptidyl leucine boronic acid, that was developed through a large-scale screening of boroncontaining PIs in the search of compounds with an increased efficacy and reduced side effects with respect to bortezomib (Kupperman et al., 2010; Chauhan et al., 2011; Offidani et al., 2014) . Since it belongs to the same chemical class of bortezomib, it is not surprising that its acts through a similar mechanism of action. In fact, proteasome subunit inhibition occurs when boric acid group forms a covalent bond with the hydroxyl group of the catalytic N-terminal threonine residue (Muz et al., J o u r n a l P r e -p r o o f 2016) . Like bortezomib, ixazomib reversibly blocks the chymotrypsin-like activity of the β5 subunit (IC 50 =3.4 nmol/L for ixazomib vs 2.7 nmol/L for bortezomib) (Chauhan et al., 2011; ) ( Figure 4C ). Noteworthy, proteasome dissociation half-life for ixazomib is relatively shorter that for bortezomib (18 min for ixazomib and 110 for bortezomib), improving its general tissue distribution, rendering this drug more "re-available" (see section 3.3.2.2.2) (Kupperman et al., 2010; . Biochemical analysis and in vitro studies showed that at high concentrations ixazomib (like bortezomib) inhibits also other proteolytic sites of 20S proteasome (Chauhan et al., 2011) ; however, the most important advancement of ixazomib with respect to bortezomib is the possibility of an oral administration; thus, ixazomib can be formulated as an ester citrate prodrug (MLN2238), which is rapidly hydrolyzed in aqueous solution (e.g., plasma) to the pharmacologically active metabolite MLN2238 with free boric acid (Kupperman et al., 2010; Chauhan et al., 2011; Okazuka and Ishida, 2018; Gupta et al., 2019) . Like bortezomib, the apoptotic activity of ixazomib is mediated by caspase 3-8-9 activation through a stabilization of the p53 pathway (see section 3.2.2 and 3.2.3) (Muz et al., 2016) . Interestingly, microRNA studies in MM cells revealed that ixazomib induces upregulation of the tumour suppresso miR33b, leading to apoptosis by blocking proto-oncogene PIM-1 (Tian et al., 2012) .

Another peptide boronate is delazomib, which reversibly inhibits CT-L subunit with a magnitude and a mechanism of action similar to bortezomib and ixazomib (Dorsey et al., 2008; Piva et al., 2008) . Moreover, like bortezomib, in vitro studies have revealed that Delazomib primary target is the inhibition of NF-kB pathway, with a consequent alteration of the expression of several NF-kB downstream effectors (Piva et al., 2008; Kubiczkova et al., 2014) .

The main representative of the PI third class is marizomib, also named Salinosporamide A, which is a natural product deriving from a sediment obligate marine actinomycete identified as Salinospora tropica (strain CNB-392) (Feling et al., 2003; Pott and Lam, 2010; Pereira et al., 2019) . It is characterized by a different non-peptide-based structure with respect to other PIs so far described.

Its unique structure displays a fused γ-lactam-β-lactone ring system containing a cyclohexenyl carbinol and chloroethyl functional groups. Marizomib is an irreversible inhibitor of all catalytic subunits of 20S, with IC 50 values ranging from the low pM to mid nM range (Feling et al., 2003; Fenical et al., 2009) , and it produces a prolonged 20S inhibition (≥72 h) (Pott et al., 2011) .

Crystallographic structure of the complex between Salinosporamide A and yeast 20S showed that the drug perfectly occupies the active sites of all three pairs of catalytic subunits of 20S (Groll et al., 2006; Fenical et al., 2009) (Figure 4D ). These findings provided a detailed understanding of the proteasome-ligand interactions at the molecular level, revealing a unique mechanism of action that J o u r n a l P r e -p r o o f renders the inhibitor irreversible. The first step of interaction is represented by the formation of a covalent ester bound between the catalytic N-terminal Thr1Oγ of each 20S subunit and the carbonyl of the β-lactone ring of the inhibitor ( Figure 5C ). β-lactone ring opening is followed by chlorine elimination, giving rise to a stable a 5-membered cyclic ether (Groll et al., 2006) . One of the main downstream effect observed after proteasome inactivation by marizomib is the inhibition of NF-κB activation, in a fashion similar to other PIs . Remarkably, as also discussed in section 3.3.2.2.3, the main advantage of marizomib is the capability to overcome the blood-brain barrier, which opened a novel therapeutic potential for this inhibitor, eliciting the research in attempt to improve its pharmacological profile (Singh et al., 2010; Park et al., 2018) .

An intriguing point on proteasome biology, which reflects on the identification of more clinically effective PIs and/or novel combination therapy, concerns the individual role and the functional meaning of different proteasome subunits. Although these crucial aspects are poorly understood, β5 subunit was initially identified as the rate limiting protease for proteasome-dependent protein turnover (Heinemeyer et al., 1997; Arendt and Hochstrasser, 1997; Groll et al., 1999) .

Consequently, PIs were designed to target β5 subunits, as it comes from the above reported chemical features of main PIs (Kubiczkova et al., 2014; Kisselev et al., 2012) . However, advanced chemical manipulations, which allowed to monitor the activity of each individual proteolytic subunit, have pointed out that, at higher concentrations, all β5-targeted PIs lose their subunit selectivity and inhibit also the β1 and/or β2 types of proteasome subunits (Kraus et al., 2015; Bruin et al., 2016) . These co-inhibition patterns differ among individual PIs and seem to be responsible for the overcoming of drug resistance observed at higher concentrations. In this respect, recent investigations have revealed that β5 and β2 co-inhibition, exclusively achieved by high levels of carfilzomib, is the most effective proteasome inhibition profile in MM (Besse et al., 2019).

Therefore, it has been proposed that a better comprehension of the significance of different coinhibitory patterns should help to understand the differential activity and toxicity observed during treatment with different PIs as well as with different doses of the same drug (Besse et al., 2019).

Moreover, these findings could provide the rationale for preclinical and clinical investigations of a novel treatment schedules. These results showed that differences on the functional proteasomeinteracting groups of the PI (i.e., epoxyketone, β-lactone, or boronate) cannot account for the observed differences in the clinical efficacy of the various drugs (reversible versus irreversible proteasome binding); differences in the PIs affinity towards the various proteasome subunits should be considered instead. The observed differences in PIs affinities are mainly due to different interactions of the specific PI side chains with each of the proteasome subunits. The chemical J o u r n a l P r e -p r o o f interpretation of the different PIs inhibition capability for the various proteasome subunits (Gozzetti et al., 2017) also explains why drugs combination is more effective than monotherapy.

In the 1990s, the reversible PI bortezomib (formerly named as PS-341) PI, was initially developed as anti-inflammatory and anti-cachectic agent. However, preclinical studies soon unravelled that bortezomib was highly effective against different tumours, in particular MM, inducing growth arrest and apoptosis and inhibiting angiogenesis (Mitch et al., 1996; Adams et al., 1998; Adams et al., 1999; Teicher et al., 1999; Hideshima et al., 2001; LeBlanc et al., 2002; Ma et al., 2003; Mitsiades et al., 2003; Sánchez-Serrano. 2006; Caravita et al., 2006) . Additionally, in vitro studies revealed that bortezomib increased in vitro tumour chemosensitivity and overcame chemoresistance to dexamethasone, doxorubicin, and melphalan, suggesting also its use in combination therapies (Hideshima et al., 2001; Mitsiades et al., 2003; Ma et al., 2003) . The overall bulk of in vitro and in vivo studies supported clinical investigations of bortezomib in patients with MM, who had received at least two prior therapies and have demonstrated disease progression after the last therapy (Caravita et al., 2006; Park et al., 2018; , leading to the first global approval of a first PI for cancer treatment.

A pivotal early phase I study investigated the maximum-tolerated dose, dose-limiting toxicity, and pharmacodynamics of bortezomib in patients with refractory hematological malignancies, showing activity against RMM (Orlowski et al., 2002) . Subsequently, a phase II study (CREST) showed the efficacy of the PI, as single agent or in combination with dexamethasone, in patients with relapsed MM after frontline therapy (Jagannath et al. 2004 ). These observations provided the rationale for a phase 2 open-label, single-arm (SUMMIT) trial, which included 202 patients with RRMM receiving at least two prior therapies, in which bortezomib (1.3 mg/m 2 ) was administered by intravenous bolus twice weekly for 2 weeks, followed by 1 week without treatment, for up to eight cycles (24 weeks). This study reported 27.7% complete or partial response rate, a median response of 12 months and manageable adverse effects . Moreover, bortezomib increased the time to progression to a higher extent (2-4 folds) compared to the last treatment patients received before entering the clinical trial. These impressive results led to the accelerated approval of bortezomib for the treatment of patients with RRMM who had received at least two prior therapies, a particularly difficult-to-treat patient population. An extended follow-up of the SUMMIT study J o u r n a l P r e -p r o o f reported a median time to progression of 13.9 months for responding patients, whereas of 1.3 months for those with progressive disease or not evaluable . The phase 3 trial, APEX, comparing bortezomib with high-dose dexamethasone for RRMM after one to three previous lines of treatment, showed a significant increased survival in patients treated with the PI (one-year survival rates of 80% versus 66%, P=0.003; the hazard ratio for overall survival (OS)= was 0.57, P=0.001) (Richardson et al., 2005) and these results led in 2005 to the regular approval to bortezomib. The superiority of bortezomib was further confirmed after an extended follow-up, in which the reported median OS was 29.8 months for bortezomib and 23.7 months for high-dose dexamethasone, despite crossover from dexamethasone to bortezomib arm (Richardson et al., 2007) . Thereafter, in 2007 FDA approved label expansion of bortezomib to include patients with impaired kidney function, without the requirement of dose adjustments).

Bortezomib was also tested in multidrug regimens, since the shift in clinical practice to a more aggressive approach, including PIs (as also discussed in Section 3.2), has improved survival outcomes (Leleu et al., 2019) . In the DOXIL-MMY-3001 phase III study the safety and efficacy of bortezomib in combination with pegylated liposomal doxorubicin were compared to those of bortezomib as a single agent in patients with RRMM who had received at least one prior treatment.

The doublet therapy was more effective than monotherapy, even though associated with a higher incidence of grade 3/4 (80 vs 64%, respectively) myelosuppression, gastrointestinal events, and hand-foot syndrome (Orlowski et al., 2007) . However, despite the significant increase in time to progression observed in the group treated with the drug combination, the final results of OS analysis after a median follow-up of 103 months indicated no significant differences between the two treatments (Orlowski et al, 2016) . The triple combination bortezomib-thalidomide-dexamethasone resulted in increased median time to progression (19.5 versus 13.8 months; hazard ratio, 0.59; P<0.001) compared to the dual combination of the immunomodulatory agent thalidomide plus dexamethasone, in patients with MM progressing or relapsing after autologous stem-cell transplantation (ASCT), as demonstrated in a phase III study (MMVAR/IFM 2005-04) (Garderet et al., 2012) . Although a direct comparison between trials is not possible, the observed time to progression was higher than that observed in the APEX trial where bortezomib was tested as single agent (6.2 months) or in the DOXIL-MMY-3001 trial where the PI was combined with liposomal doxorubicin (9.3 month). The addition of bortezomib to thalidomide-dexamethasone was associated with a substantial increase of cumulative, dose-related grade 3 peripheral sensory neuropathy (Garderet et al., 2012) . Since thalidomide is also neurotoxic, in the triple combination this agent was replaced by lenalidomide, which is a better-tolerated analogue. Indeed, a phase II trial demonstrated a similar median time to progression of 19.5 months, but a markedly lower rate of J o u r n a l P r e -p r o o f grade 3 peripheral neuropathy compared to the triple combination including thalidomide (2% versus 29%) . The current guidelines recommend the triple combination of bortezomib, lenalidomide and dexamethasone as a preferred salvage regimen for previously treated multiple myeloma and as first-line therapy irrespective of transplantation eligibility (National In the phase 3 CASTOR trial, the addition of darutumumab resulted in significantly longer PFS (median PFS 16.7 vs. 7.1 months, HR = 0.31) compared to bortezomib plus dexamethasone, but it was associated to infusion-related reactions and higher rates of thrombocytopenia and neutropenia .

Panabinostat was the first pan-HDAC inhibitor approved to treat MM, which acts via epigenetic modification and inhibition of the aggresome pathway (i.e. a proteasome-independent pathway that eliminates misfolded proteins). The approval for RRMM was based on the results from the pivotal A phase 2 study has also investigated the combination of bortezomib plus dexamethasone with the immunomodulatory agent elotuzumab, a monoclonal antibody against SLAMF7 (signalling lymphocytic activation molecule F7), reporting encouraging results (median PFS 9.7 vs. 6.9 months) . Based on the results of this study, the National Comprehensive J o u r n a l P r e -p r o o f Once bortezomib efficacy was established for RRMM in the early 2000s, attention turned to patients with newly diagnosed disease (NDMM), in whom its efficacy was tested with or without dexamethasone, showing that the combined treatment was associated with improved response rate without additional severe toxicities compared to PI monotherapy (Jagannath et al., 2005; Harousseau et al., 2010; Okazuka and Ishida, 2018) . In 2008, the key phase 3 trials VISTA led to the approval of bortezomib, in combination with melphalan and prednisone, by FDA for previously untreated MM and by EMA for previously untreated MM not eligible for high dose chemotherapy and stem-cell transplantation (SCT) . Melphalan plus prednisone, was the standard of care for NDMM patients over 65 years old, being instead high-dose chemotherapy followed by SCT the preferred treatment for patients under the age of 65 years (Barlogie et al., 1997; Alexanian et al., 1969; . In the VISTA trial, 682 patients were with previously untreated MM, eligible for high dose chemotherapy followed by SCT. Thereafter, the bortezomib-lenalinomide-dexamethasone triplet regimen has become one of the standard induction therapies before SCT (Okazuka and Ishida, 2018) . In two different phase 2 studies (i.e., IFM and IFM/DFCI 2009) this therapeutic regimen was tested in patients with NDMM eligible for SCT as induction therapy, and as induction and consolidation therapy, respectively, with encouraging results in terms of PFS (Richardson et al., 2010; Roussel et al., 2014) . Additionally, results from a phase 3 clinical trial demonstrated that the lenalidomide-containing triplet therapy (RVD) followed by high-dose chemotherapy plus SCT was associated with significantly longer PFS than the RVD therapy alone, even though OS did not differ significantly between the two approaches (Attal et al., 2017) . A more recent study also confirmed the efficacy of the RVD regimen in the pre-transplant induction therapy so that it has to be considered as a standard of care Initially, intravenous injection was the standard administration route for bortezomib. Thereafter, a large randomized phase 3 clinical trial, compared the efficacy and safety of subcutaneous versus intravenous treatment, at the approved 1.3 mg/ml dose and twice per week schedule in patients with RRMM, showing that subcutaneous administration induced similar effect in terms of overall response rate, but with improved tolerability and reduction of the incidence of peripheral neuropathy. Thus, currently subcutaneous injection is the preferred method of bortezomib administration, since this route is also more convenient for patients (Moreau et al., 2011; Arnulf et al. 2012) .

Common adverse effects associated with bortezomib administration are fatigue, gastrointestinal toxicity, trombocytopenia, anorexia, and peripheral neuropathy (i.e., hyperesthesia, hypoesthesia, neuropathic pain, weakness), which is one of the most important complications that negatively affects the patient's quality life and daily activity (Seval and Beksac, 2018) . Peripheral neuropathy has been regarded as an off-target effect, since it is due to inhibition of HtrA2/Omi, a serine protease involved in neuronal survival with potency near or equivalent to that for the proteasome (Arastu-Kapur 2011; Park et al., 2018) .

Moreover, other adverse events described so far are: (i) tumour lysis syndrome (Sanagawa et al., 2020) ; (ii) cardiovascular toxicities (Enrico et al., 2007; Grandin et al., 2015) , (iii) acute interstitial nephritis and rarely (iv) a severe syndrome of inappropriate anti-diuresis (SIAD) (Peng et al., 2017;  J o u r n a l P r e -p r o o f Cheungpasitporn et al., 2015) . Furthermore, treatment with bortezomib is associated with an increased risk of Varicella Zoster Virus (VZV) infection, and a continuous prophylaxis with antiviral agents, such as acyclovir and valacyclovir, is recommended (Aoki et al., 2011; Chanan-Khan et al., 2008; Teh et al., 2016; .

The pharmacokinetics of bortezomib is poorly documented mostly due to analytical difficulties (Leveque et al., 2007) . Two different studies on patients with prostate cancer and MM suggested that its kinetic profile is characterized by a large distribution volume (Vd), 721-1270L, high systemic clearance, ranging from 1095 mL/min to 1866 mL/min, and terminal half-life ranging between 10 h and 31 h (calculated over a 24 h period) (Papandreou et al., 2004; Leveque et al., 2007) .

For what concerns bortezomib clearance, the drug is converted into inactive de-boronated metabolites by different cytocrome P450 enzymes (CYPs) (e.g., 1A2, 2C9, 2C19, 2D6, and 3A4) (Uttamsingh et al., 2005; Pekol et al., 2005) , as also confirmed by studies in which the coadministration of ketoconazole, a CYP3A4 inhibitor, and rifampicin, a CYP3A4 inducer, increased and decreased patients' exposure to bortezomib, respectively (Venkatakrishnan et al., 2009; Hellmann et al., 2011) . Since bortezomib undergoes oxidative metabolism in the liver, a study was carried out on whether patients with a reduced hepatic functionality require dose adjustment investigation (Venner et al., 2012; Mikhael et al., 2012; Palladini et al., 2014; Kastritis et al., 2019; . In recent years, the introduction of bortezomib has had a great impact in the cure of Mantle cell Lymphoma (MCL), a non-Hodgkin lymphoma with a short remission duration to standard therapies, and a median OS of approximately 6-7 years (Banks et al., 1992; Fisher et al., 1995; Teodorovic et al., 1995; Weisenburger et al.,2000; Vose et al., 2017) . Therefore, there is a great need of therapeutic strategies directed against novel molecular targets. The chromosomal translocation t(11;14)-(q13;q32) is the molecular hallmark of MCL, resulting in overexpression of cyclin D1, which is not typically expressed in normal lymphocytes (Vose, 2017) , and the constitutive activation of NF-kB, which also plays a key role in MCL growth and survival (by controlling cyclin D1 expression, as reported in a previous section (see Section 3.2.1) (Rosenberg et al., 1991 , Pham et al., 2003 . Therefore, proteasome inhibition has been envisaged as an achievable therapeutic strategy, which was confirmed by in vitro studies showing that NF-kB inhibition mediated by bortezomib leads to cell cycle arrest and apoptosis in MCL cells (Pham et al., 2003) .

Based on preclinical studies and a phase 1 trial in patients with refractory hematologic malignancies, including, besides MM (see above), MCL and follicular lymphomas (Orlowski et al., 2002) , achieved an objective response), and bortezomib plus cyclophosphamide-doxorubicin-vincristineprednisone (CHOP) (OS= 36.6 for patients treated with bortezomib plus CHOP and 11.6 months for J o u r n a l P r e -p r o o f whom treated with CHOP alone) (Weigert et al., 2009; Orciuolo et al., 2010; Agathocleous et al., 2010; Furtado et al., 2015; Kouroukis et al., 2011; Lamm et al., 2011; Friederberg et al., 2011) .

Bortezomib as single agent was tested also in previously untreated MCL, demonstrating clinical activity (Belch et al., 2007; . However, more promising results were obtained when bortezomib was combined with rituximab-CHOP (R-CHOP), as demonstrated by: (i)

phase 1/2 studies on previously untreated patients with MCL (Ruan et al., 2011; Till et al., 2016; Vos et al., 2017) ; (ii) a phase 2 study on patients with newly diagnosed MCL, who received also bortezomib as maintenance therapy (Ruan et al., 2011; Till et al., 2016) . These trials demonstrated that the combination of R-CHOP with bortezomib followed by bortezomib maintenance improves PFS, as compared with R-CHOP alone, with acceptable toxicity, suggesting further investigation (Ruan et al., 2011; Till et al., 2016) . In a large, randomized phase 3 trial, 487 patients with untreated, newly diagnosed MCL, who were not eligible for transplantation, were randomly assigned to two groups, one receiving R-CHOP and a modified R-CHOP regimen with bortezomib 

On the basis of bortezomib success in haematological malignancies, its potential application in the treatment of solid tumours has been explored (Roeten et al., 2017) . A large amount of data has been collected in vitro and in vivo aiming at characterizing the possible activity of bortezomib in different models, such as pancreatic and breast cancers, hepatocellular and anaplastic thyroid carcinoma, with contradictory results (Chen et al., Roeten et al., 2018) .

One of the most promising strategy is the combination of bortezomib with radiotherapy, which results in synergistic effects as a consequence of the bortezomib-induced cell accumulation at the Table 1 ), revealing, as for preclinical studies, conflicting results. Due to their poor prognosis, two types of solid tumours have been mainly investigated, namely: (iv) resistance to apoptosis induction, even though the exact role of each one of these mechanisms needs to be further investigated. Interestingly, to overcome the poor penetration of bortezomib in solid tumours, an alternative strategy currently investigated consists in a delivery system based on nanoparticles or micelle formulation, as also studied for other PIs Ao et al., 2015; Coelho et al., 2016) .

The strategy of proteasome activity inhibition and the introduction of bortezomib in clinical practice have dramatically changed the battle against MM, even though it was immediately evident that this J o u r n a l P r e -p r o o f drug suffers from several drawbacks that needed to be overcome. For example, (i) mutation in the β5 subunits, (ii) induction of drug efflux from cells and (iii) activation of signaling cascades promoting cell survival are all resistance mechanisms that have been identified in bortezomibresistant cell lines (Oerlemans et al., 2008; Sherman and Li, 2020) . Therefore, second-generation

PIs have been designed and tested, including carfilzomib, ixazomib, delanozomib, oprozomib and marizomib, whose properties will be discussed in the next sections. Particular emphasis will be given to carfilzomib and ixazomib, which are both FDA-and EMA-approved.

As mentioned in the previous section, two main limitation in the clinical use of bortezomib are (i) the extent of proteasome inhibition and (ii) proteasome recovery after inhibition (Deshaies et al., 2014) . These issues stimulated an intense research, which culminated with the discovery of the natural compound epoxomicin, a covalent and irreversible inhibitor of the β5 subunit (Hanada et al., 1992; Meng et al., 1999; Myung et al., 2001; Deshaies et al., 2014) , which was isolated by an unidentified Actinomycetes strain (see also Section 3.3.1.2). Importantly, epoxomicin owned unprecedented and exceptional selectivity for proteasome with respect to the proteasome inhibitors already available Kim and Crews, 2013) . This high selectivity was supposed to guarantee more physiological tolerability, and a more favourable pharmacological profile than bortezomib. Moreover, the ability to irreversibly bind the β5 subunit implies that the only way to recover proteasome activity is the induction of novel synthesis of functional proteasome particles and dexamethasone as supplemental application for the treatment of patients with MM, who had received one to three prior lines of therapy Stewart et al., 2016) . In this trials, 792 patients with RRMM were randomly assigned in a 1:1 ratio to the carfilzomib group, in which the drug was part of a triple combination therapy with lenalidomide and dexamethasone, or to the lenalidomide plus dexamethasone control group . Carfilzomib was administrated for 18 cycles, at a starting dose of 20 mg/m 2 on the first cycle with subsequent escalation to reach the target dose of 27 mg/m 2 in the following cycles. The addition of carfilzomib significantly increased the median PFS as compared with lenalidomide and dexamethasone alone (26.3 months vs. 17.6 months; HR for progression or death, 0.69; P=0.0001). Furthermore, the J o u r n a l P r e -p r o o f triplet therapy showed a favourable risk-benefit profile and improved the health-related quality of life of RRMM patients Stewart et al., 2016) . Moreover, in 2016 FDA extended carfilzomib approval, in combination with dexamethasone, to patients with RRMM, who had received one to three lines of therapy, on the basis of results of phase 3 randomized, open label, ENDEAVOR study (NCT01568866). In this head-to head comparative study of bortezomib and carfilzomib, 929 patients with RMM were randomly assigned to receive carfilzomib plus low-dose dexamethasone or bortezomib plus low-dose dexamethasone . In this study, carfilzomib regimen was 27 mg/m 2 in the first cycle and 56 mg/m 2 thereafter, infused over 30 minutes, which is the maximum tolerated dose of carfilzomib tested in combination with dexamethasone in phase 1/2 clinical trials (Papadopulos et al., 2015; . The primary endpoint of the trial was PFS that was reported to be longer for the carfilzomib group, as compared to the bortezomib one (18.7 versus 9.4 months, HR=0.53; P<0.0001) . In another interim analysis aimed at comparing the OS between the two PI, it has been shown that patients treated with carfilzomib had a statistically significant and clinically meaningful improvement in OS than those treated with bortezomib ( with RRMM, who had received at least two (but no more than three) prior therapies (including bortezomib and an immunomodulatory drug), were assigned to receive a 30-minute infusion of once-weekly (70 mg/m 2 ) carfilzomib vs a 10-minute infusion of twice-weekly (27 mg/m 2 ). All patients also received the same dose of dexamethasone . The primary endpoint of the trial, PFS, was 11.2 months for the once-weekly regimen versus 7.6 months for the twiceweekly one (HR=0.69; P=0.0014). The ORR in patients treated with the once-weekly regimen was 62.9% versus 40.8 % for those treated with twice-weekly (p<0.0001) Moreau et al., 2020) . Thus, the once-weekly carfilzomib was safe and more effective as compared to the twice weekly schedule.

As mentioned above, early phase 1/2 trials suggested that carfilzomib in combination with other agents, such as melphalan and prednisone (Moreaue et al., 2015), lenalidomide and low-dose dexamethasone (Jakubowiak et al., 2013) , or thalidomide and low-dose dexamethasone (Wester et al., 2019) , could be a therapeutic opportunity also for NDMM patients, although the outcomes and the related adverse events are still not convincing. Carfilzomib seems to have a distinct pattern of adverse effects with respect to bortezomib. In fact, the rate of peripheral neuropathy is lower than J o u r n a l P r e -p r o o f for bortezomib, whereas some patients are affected by cardiovascular complication, such as hypertension and heart failure, rendering the ecocardiography assessment advisable before the onset of treatment. Additionally, unlike bortezomib, carfilzomib can lead to renal failure Chari et al., 2014; Korde et al., 2015; Manasanch and Orlowski, 2017) . Adverse events in common with bortezomib are fatigue, anemia, nausea and thrombocytopenia .

Currently, a great number of clinical studies on carfilzomib are listed in clinical trial.com. Besides MM, carfilzomib is evaluated in clinical trials for solid tumours, including lung, refractory renal, and metastatic prostate cancers (Table 2 ). However, like bortezomib, its therapeutic potential is limited by the low distribution within the tumour mass, thus requiring very high and toxic doses to elicit a response (Huang et al., 2014; Grigoreva et al., 2015; Johnson, 2015) . Quach et al., 2017) . This is of particular relevance, taking into account that renal insufficiency is a common and often severe complication occurring in MM patients.

Both bortezomib and carfilzomib require parental administration, and are associated to the development of specific toxic effects (see Sections 3.3.1.1 and 3.3.2.1a), mainly peripheral neuropathy and cardiovascular adverse events (carfilzomib) Dimopoulus et al., 2011; Richardsone et al., 2012; Dimopoulos et al., 2017; Waxman et al., 2018) . In fact, although the weekly dosing and subcutaneous administration of bortezomib have attenuated the risk of peripheral neuropathy with bortezomib, this adverse effect is still an important concern. Moreover, bortezomib has a limited tissue distribution, due to its slow dissociation rate from red blood cells. Thus, development of an orally available PIs with improved pharmacokinetics properties and better tolerability profile was required (Gupta et al., 2019) . Accordingly, ixazomib J o u r n a l P r e -p r o o f (Ninlaro) represents the first orally administered PI approved for clinical use by FDA and EMA.

This agent is a reversible inhibitor that preferentially binds the β5 site of the 20S proteasome. In vitro and in vivo preclinical studies reported for ixazomib a therapeutic efficacy greater than for bortezomib in different cancer models, including MM, as well as improved pharmacokinetics, pharmacodynamics, and antitumour activity in xenograft models. In particular, in MM xenograft models, mice treated with ixazomib presented a significant longer survival time than those treated with bortezomib (Chauhan et al., 2005; Kupperman et al., 2010; Chauhan et al., 2011; . In addition, ixazomib induced apoptosis in MM cells resistant to bortezomib without affecting the viability of normal cells, suggesting a potential efficacy in patients with disease relapse after treatment with bortezomib-containing regimens (Chauhan et al., 2011) . Therefore, based on the encouraging results observed in these preclincal studies, together with its more convenient oral administration, ixazomib rapidly advanced in phase 1 clinical trials on RRMM to evaluate its safety and tolerability, as single-agent administered once-weekly (NCT00963820) or twice-weekly (NCT00932698) (Kumar et al., 2014 a; . In the once-weekly dosing study, the maximum tolerated dose was determined to be 2.97 mg/m 2 , whereas in the twiceweekly dosing was 2 mg/m 2 . Overall, ixazomib was generally well tolerated; no severe neuropathy was reported and most of the observed toxicities were manageable. These studies also indicated that ixazomib absorption is rapid, with a maximum plasma concentration at approximatively 1 h postdose. After multiple dosing, the terminal half-life was 3.3-7.4 days and 3.6-11.3 days in the onceweekly and twice-weekly regimes, respectively. The ORR were 18% and 15% for one weekly and twice-weekly treatments, respectively, supporting the use of both schedules (Kumar et al., 2014 a; . The efficacy of ixazomib as a single agent (5.5 mg/m 2 weekly for 3-4 weeks) was confirmed in the first part of a phase 2 trial recruiting 33 patients with relapsed MM who were PI naïve or previously exposed to bortezomib but were not refractory to this agent (NCT01415882) (Kumar et al., 2015) . Moreover, in a second phase of this trial the efficacy and tolerability of ixazomib were evaluated in combination with dexamethasone in patients showing lack of adequate response or disease progression. The ORR was 34% and the main toxic effects observed were nausea, thrombocytopenia and fatigue that were in line with the ixazomib toxicity profile (Kumar et al., 2015) . The efficacy of the ixazomib (weekly doses of 4 or 5.5 mg/m 2 ) and

dexamethasone combination was further investigated in another phase 2 trial recruiting patients with MM that had relapsed after at least 1 previous therapy but not refractory to bortezomib. The results of this study revealed that the ixazomib-dexamethasone doublet had promising efficacy and acceptable tolerability (ORR = 31% and 51% with 4.0 mg and 5.5 mg, respectively). However, the J o u r n a l P r e -p r o o f combination with the higher dose of ixazomib was more toxic albeit, indicating the potential requirement of dose reductions to attenuate adverse effects .

In preclinical studies ixazomib was shown to exert synergistic effects with lenalidomide, and the results of these studies provided the rationale for the clinical testing of the PI with lenalidomide plus desamethasone (Chauhan et al., 2010) . The clinical efficacy and manageability of adverse events reported in early trials , were confirmed in the phase 3, randomized, double-blind, placebo-controlled TOURMALINE-MM1 trial (NCT01564537), whose results led to FDA (2015) and EMA (2016) approval of the triplet regimen combining ixazomib with lenalidomide and dexamethasone in MM patients, who had received at least one prior therapy . In this trial, 722 patients, who had RMM or RRMM, were randomly assigned to receive ixazomib plus lenalidomide-dexamethasone (ixazomib group) or placebo plus lenalidomide-dexamethasone (placebo group). Interim results demonstrated that the addition of ixazomib significantly prolonged PFS compared to the control group (median 20.6 vs. 14.7 months; HR= 0.74; P=0.01). Importantly, a benefit in terms of PFS was observed with ixazomib regimen in all patient subgroups, including subjects with high-risk cytogenetic abnormalities (del(17p), t(4;14), and/or t(14;16)), who are known to be burdened by a very severe prognosis Avet-Loiseau et al., 2016) . An arm of the TOURMALINE-MM1 trial also included patients previously treated with prior PI therapy and prior thalidomide/ lenalidomide combination therapy;

results demonstrated a substantial clinical benefit in terms of prolonged PFS with the ixazomiblenalidomide/dexamethasone triplet regardless of prior administered therapy . , 2017) . Importantly, the combination of ixazomib with the lenalidomidedexamethasone regimen was associated with a limited additional toxicity, and had no adverse impact on patient-reported quality of life. Commonly reported grade ≥3 adverse events with ixazomib include gastrointestinal symptoms, rash, thrombocytopenia, and arrhythmia Hou et al., 2017; Leleu et al., 2018; Hari et al., 2018) . For what concerns the peripheral

The triplet regimen ixazomib-lenalidomide-dexamethasone followed, when feasible, by singleagent ixazomib as maintenance therapy, was investigated also in patients with NDMM in different trials. In in a phase 1/2 study (NCT01217957), the combination therapy was well tolerated and associated with high ORR (92%) (Kumar et al., 2014) . Furthermore, analysis of the long-term efficacy and safety of this regimen, confirmed that ixazomib-lenalidomide-dexamethasone followed by ixazomib maintenance was highly active and caused manageable toxicity in this clinical setting.

In particular, out of 65 enrolled patients, 23 patients discontinued induction for SCT, whereas in the remaining 42 patients, the ORR was 80%, including 63% very good partial response and 32% complete responses; these data underscore the feasibility of long-term maintenance treatment with single-agent ixazomib . Furthermore, in NDMM patients, a phase 1/2 doseescalation study investigated the all-oral ixazomib-melphalan-prednisone regimen, followed by single-agent ixazomib maintenance, in transplant ineligible patients with encouraging results (San-Miguel et al., 2018) . Recently, the TOURMALINE-MM3 trial (NCT02181413) investigated the ixazomib suitability versus placebo as a maintenance therapy in NDMM to delay disease progression and prolong patients' survival following ASCT. The results of this study revealed that ixazomib induced a 28% reduction in the risk of progression or death compared to placebo (median PFS 26.5 months vs 21.3 months; HR= 0.72; P=0.0023), thus representing an additional therapeutic option for these patients . Promising results come also from an ongoing phase 3 trial where ixazomib is administered in patients with RMM as post-ASCT maintenance strategy (Striha et al., 2018) .

Several studies are currently investigating the activity of ixazomib in patients with immunoglobulin light chain (AL) amyloidosis, Waldestrom Macroglobulinemia, bone plasmocytome and other nonhaematological malignancies (Smith et al., 2015; Smolewski and Rydygier, 2019; clinical trials.gov) . Until now the best results were obtained in a phase 1/2 study which evaluated the safety, tolerability, and preliminary efficacy of ixazomib in patients with relapsed/refractory AL amyloidosis, paving the road to a phase 3 study which is currently ongoing (NCT01659658) (Sanchorawala et al., 2017; Smolewski and Rydygier, 2019) .

Using all collected clinical data, ixazomib pharmacokinetics was characterized by an absolute oral bioavailability of 58%, terminal long half-life of 9.5 days, large distribution volume of 543 L, and systemic clearance of approximately 1.86 L/h Park et al., 2018; . The faster dissociation rate of ixazomib compared to bortezomib, which allows it to associate and dissociate consecutively with more than one proteasome particle, likely contributes to the improved drug distribution into tissues (Kupperman et al., 2010) . Moreover, it has been shown J o u r n a l P r e -p r o o f that plasma exposure increases linearly with higher administered dose, and no dose adjustment is required on the basis of race, age, sex, body weight, mild-moderate renal impairment, and mild hepatic impairment Gupta et al., 2016; Gupta et al., 2019) . At clinical doses, ixazomib is mainly metabolized by non-CYP enzymes; in fact, no significant effect on its pharmacokinetics has been reported after the concomitant administration of CYP3A inhibitors, such as ketoconazole and clarithromycin, in patients with advanced solid tumours and lymphoma (NCT01454076) . However, the concomitant administration of the CYP3A-inducer rifampin causes a clinically relevant reduction in ixazomib activity, supporting the advice to avoid this combined treatment schedule, and underlying the complexity of ixazomib metabolism Gupta et al., 2019) . In all clinical trials so far described, ixazomib was administered on an empty stomach (Gupta et al., 2019) . However, since the absorption and metabolism of an oral drug can change with food, according to the US regulatory guidance (Singh et al., 2004 ; United States Food and Drug Administration), a phase 1 study in adult patients with advanced solid tumours or lymphoma was carried out to evaluate whether pharmacokinetics of ixazomib might be altered when administered after a high-calorie, high-fat meal (Gupta et al., 2016; Gupta et al., 2019) . Currently, 139 clinical studies on ixazomib are reported in clincaltrials.gov, also including the studies on solid tumors ( Table 2 ). The results of this study showed that a high-fat meal reduces the rate and extent of absorption of ixazomib, supporting its administration on empty stomach, at least 1 hour before or at least 2 hours after food intake. These recommendations are inserted in the ixazomib prescribing information (Gupta et al., 2016; Gupta et al., 2019) .

To overcome the clinical limitations of FDA/EMA approved PIs, a number of novel compounds have been identified over the last years. However, the only three drugs currently under evaluation in clinical trials are: oprozomib (PER-047 and ONX 0912); marizomib (NPI-0052, salinosporamide A) and delanzomib (CEP-18770) ( Table 3) .

Oprozomib is an oral drug designed to improve the absorption rate, dosing flexibility, and to overcome two established bortezomib-resistance mechanisms, such as mutations in the proteasome β5 subunit, and drug efflux mediated by ATP-binding cassette transporters (Zhou et al., 2009; Verbrugge et al., 2012) . Preclinical studies showed that oprozomib has an antitumour activity comparable to that of carfilzomib, stimulating early clinical investigation (Chauhan et al., Park et al., 2018) . Some phase 1b/2 trials have evaluated oprozomib efficacy and safety profile as The pharmacokinetic profile of oprozomib was first investigated in preclinical models: the drug was rapidly absorbed (2-3 minutes) in duodenum and jejunum with an estimated absolute oral bioavailability of approximately 39% (Zhou et al., 2009; Park et al., 2018) . Moreover, phase I studies revealed a plasma half-life of about 1 h and a clearance that exceeded the hepatic blood flow, indicating extra-hepatic contribution to its metabolism. Accordingly, the epoxide hydrolase, which seems to be the primary enzyme involved in oprozomib metabolism, is expressed in many other tissues beyond the liver (Fang et al., 2015; . Therefore, though developed to J o u r n a l P r e -p r o o f improve carfilzomib pharmacokinetics properties, oprozomib still displays a high systemic clearance and a short half-life Fang et al., 2015) .

Marizomib is different from the structural point of view with respect to other PIs, and this translates into a different mechanism of proteasome inhibition, efficacy and toxicity profile (Gozzetti et al., 2017) . In vitro and in vivo studies demonstrated that marizomib induces apoptosis in MM and other haematological and solid malignancies with a lower toxicity compared to bortezomib (Ruiz et al., 2006; Pott et al., 2011) . Importantly, marizomib induced apoptosis even in tumour cells from MM patients relapsing after various prior therapies including bortezomib and/or thalidomide (Pott et al., 2011; Chauhan et al., 2005; Singh et al., 2010) . Early phase clinical trials testing different treatment schedules of the PI as single agent in patients with advanced malignancies, reported marizomib activity mainly in patients with RRMM. Remarkably, marizomib did not exhibit severe peripheral neuropathy, warranting further evaluation Levin et al., 2016) . The most important adverse events observed in the phase 1 trials were fatigue, nausea, diarrhoea, and infusion site pain . In accordance with preclinical models in which marizomib was found to synergistically act with immunomodulatory agents (Chauhan et al., Das et al., 2015) , a phase 1 clinical trial demonstrated that the triplet combination of marizomib, pomalidomide and low-dose dexamethasone was well tolerated and endowed with promising activity in heavily pretreated, high-risk RRMM patients, without increasing the incidence of adverse events . As mentioned in previous sections, PIs are relatively ineffective in treating solid tumours (Dou & Zonder, 2014) . Thanks to its more lipophilic structure, an additional differential feature of marizomib compared to other PIs is the ability to cross the blood brain barrier in different species.

Accordingly, preclinical studies demonstrated that oral administration of marizomib inhibits proteasome activity in the brain, and displays a greater activity than bortezomib in a range of solid tumour xenograft models (Pott et al., 2011; Shabaneh et al., 2013; Di et al., 2016) . In fact, marizomib was found to induce apoptosis in glioma cells, with minimal toxic effect on normal neurons Manton et al., 2016) . Based on these studies, this PI is currently evaluated in a clinical trial (NCT03345095) for treating newly diagnosed glioblastoma, the most common aggressive malignant primary brain tumour in adults, having a median survival of about 12 months, after debulking surgery and radiotherapy (Weller et al., 2019) .

The pharmacokinetics profile of intravenously administered marizomib was investigated in a phase 1 clinical trial on patients with advanced solid malignancies, indicating a short half-life (lower than 30 minutes), rapid clearance (0.9-22 L/min), and a large volume of distribution (15-416 L) . Although the involvement of extra-hepatic clearance in the overall J o u r n a l P r e -p r o o f Journal Pre-proof marizomib elimination has been proposed, detailed studies on excretion, metabolism, and in general pharmacokinetics-pharmacodinamic profiles are not available (Pott et al., 2011; Harrison et al. 2016; Park et al., 2018) .

Delanzomib is an oral PI that in vitro has shown significant activity on MM and a panel of solid tumours. Furthermore, both intravenous and oral administration resulted in complete tumour regression in MM xenograft models, and increased mice survival in a systemic model of human MM (Piva et al., 2008) . Moreover, administration of delanzomib in combination with other conventional anti-MM therapies, such as melphalan plus bortezomib and dexamethasone plus lenalidomide, was more effective than treatment with either agent alone (Sanchez et al., 2010; Sanchez et al., 2012) . Nevertheless, the results of early phase trials were not so encouraging (Gallerani et al., 2013; Vogl et al., 2017) . In a phase 1 trial, delanzomib showed a linear plasma pharmacokinetics profile, lack of peripheral neuropathy, but a very high incidence of severe skin toxicity in 53% of patients (Gallerani et al., 2013) . In a second multicentre phase 1/2 study delanzomib as single-agent was investigated in patients with RRMM, but no efficacy was reported, whereas severe adverse events, such as rash and thrombocytopenia were reported. Thus, development of delanzomib for myeloma was discontinued (Vogl et al., 2017) . To date, it is not clear the rationale of different clinical and preclinical investigations (Park et al., 2018) .

Beside the inhibitors, described in Section 3.3, which switch off directly the enzymatic activity of the proteasome by locking out the active site(s) through a chemical bond with specific residues, new class(es) of molecules is/are emerging, which act(s) instead as a modulator(s) of the proteasome activity rather than directly inhibiting it. Obviously, they are not alternative to other inhibitors, which often inhibit more efficiently the proteasome enzymatic action, but they rather affect the UPS activity in a variable extent and fashion, interfering at a different level, such as the interaction with

Porphyrins are an old class of antitumour agents which is now again at the center of renewed scientific interests for the possible role as multifunctional (antitumoural) drug. They are organic heterocyclic macrocycles with an extended π system that on one hand makes them highly hydrophobic and, on the other hand, provides porphyrins with a remarkably high extinction coefficient together with additional photo-physical properties. The latter properties make them wellsuited to accomplish, for example, both clinical phototherapy (PDT) and cancer imaging, rendering them suitable to be employed in a multi-tasking role as theranostic tools (Tsolekile et al.2019 ).

Apparently, their poor aqueous solubility might represent a major restriction to their application in proteasome, encouraging to screen the ability of porphyrins to inhibit the proteasome activity.

The first evidence that porphyrins, in particular hemin, could reduce "the protein degradation ATP dependent", not yet known as proteasome, dates back to 40 years ago (J.D. Etlinger and A.

J o u r n a l P r e -p r o o f Goldberg, 1980) . Afterwards, many reports showed that hemin effect on ubiquitin-dependent proteolysis is not restricted to erythroid cells but hemin is a negative UPS modulator in all eukaryotic cells (Haas and Rose, 1981; Vierstra and Sullivan, 1988 ).

The first molecular investigation on purified 20S has demonstrated that micromolar amounts of cationic porphyrins inhibit reversibly all three main protease activities of the proteasome (Santoro et al., 2012) . Quite interestingly, porphyrins activity is finely modulated (tuned) both by the nature and reciprocal topology of peripheral substituents and by the stereochemistry of the macrocyclic ring center. Thus, the inhibitory efficiency of the cationic macrocycles increases with the number of positive substituents in the meso position. As far as the porphyrin core is concerned, it is evident from the experimental data that among the various metallo-derivatives, the most active ones are those with no axial ligands, the activity decreasing going from penta-to hexa-coordinated metals. In particular, the naked cationic porphyrins are the most active ones, indicating that the molecule should be flat in order to interact effectively with the proteasome. Interestingly, thanks to their high extinction coefficient these molecules are "visible inhibitors", and in this sense they behave as very efficient spectroscopic "probes" for UV-Vis stopped-flow kinetic analysis. The latter studies, combined with NMR and computational study, helped in defining the tetra-cationic H 2 T 4 as a competitive inhibitor which binds the gate area on the α-ring, hindering the substrate access to catalytic chamber .

Starting from the first evidence, an accurate kinetic and computational analysis of the surface of the α-subunit ring revealed then that the positive charges play a critical role in the inhibition of the 20S,

showing that cationic porphyrins may act as tuneable gatekeepers of the 20S (see Fig. 9 in . Indeed, the α-ring represents a receptor-like region physiologically involved in ionic interactions with canonical RPs; as a matter of fact, the regular arrangement of aminoacidic residues in these surfaces has been found to represent a sort of electrostatic code, exploited by the 19S, and regulating the gating phenomena (see Sections 2.2.1 and 2.3.1). The charges of porphyrins represent the keys able to interfere with this "electrostatic code", and, depending on their spatial distribution, a high variety of binding modes and inhibition mechanisms have been observed (Di Dato et al., 2017) . Furthermore, some functional effects, characterized by cooperative phenomena, are the resulting of conformational rearrangements that can reverberate onto the β5 subunit (Arba M, et al., 2018) . Finally, additional binding modes involve interactions with both the α-and β-rings regions, acting directly on the β5 catalytic subunit.

In conclusion, porphyrins are excellent candidates for multi-tasking biological active molecules. As an example, quite recently it was shown (Vallelian et al., 2015) that high levels of intracellular 

The use of metal complexes as anticancer drugs has been adopted since early 1960s with the discovery and development of cisplatin and its derivatives (Alderden et al., 2006) . The first metal complexes used as anticancer agents were designed to interact with the cancerous cell DNA, inducing apoptosis of cancer cells, but it has been also demonstrated that they can alter the cellular redox chemistry through binding of the metal or ligand redox centers to biomolecules involved in cellular redox pathway. Since tumor cells have a more reducing environment than normal cells (due to the accelerated metabolic activity, high rates of cell growth and proliferation), selectivity can be reached by using metal complexes which contain redox active metal ions; these are then reduced in the reducing environment of the cancer cell and metal complex drugs become activated. Therefore, the reduced metal ion (Co 2+ , Pt 2+ , Fe 2+ , Cu + , etc.) exerts its anticancer activity with a marked selectivity for cancer cells, as the unique ability of metal complexes to undergo redox activation processes involves both metal and ligand redox centers and it can be tuned to specific potentials (P. Zhang & Sadler, 2017) . However, the idea of using metal complexes to disrupt proteasome activity in order to have an anticancer effect is relatively recent (Shagufta & Ahmad, 2020) . Disulfiram was the first metal complex containing copper capable of inducing apoptosis in cancer cells by inhibiting proteasome activity (Chen et al., 2006) . It was later demonstrated that disulfiram rapidly converts in vivo to its reduced metabolite diethyldithiocarbamate (DDTC) before exerting its anticancer activity (Pang et al., 2007) . Interestingly, in most cases the presence of the metal ion turned out to be fundamental to have proteasome activity inhibition, since the non-metallated ligand has been demonstrated to be ineffective for this purpose. For example, the asymmetric ligands, containing pyridine and 4,6-substituted phenol moieties alone, do not have any influence on proteasome activity (Shakya et al., 2006) ; conversely, the copper (II) chloride salt of this compound inhibited the proteasome activity in cell free conditions and, for this reason, it has been proposed that the copper complex works as a carrier to cross the cell membrane. Such an assumption would However, elucidating the molecular mechanisms by which copper ions are able to inhibit proteasome activity is a very challenging task, due to the very complex cellular environment and the difficulty in monitoring the fate of intracellular copper ions (Satriano et al., 2013) . Indeed, it has been reported in cell-free conditions that Cu (II) ions promote conformational changes associated to an impaired channel gating, without catalyzing redox reactions nor disrupting the assembly of the 20S proteasome . On the contrary, HeLa cells, grown in a Cu (II)supplemented medium, exhibit a decreased proteasome activity, which was then restored in the presence of an antioxidant. For this reason, it has been proposed that, although the Cu(II)-inhibited 20S activities may be associated to proteasome conformational changes, favoring the closed state of the core particle, other effects may occur, such as ROS-mediated proteasome flooding and disassembly of the 26S proteasome into its 20S and 19S components.

Beside copper, other metal ions have been used in complex with various ligands to inhibit proteasome activity. For example, cadmium, though carcinogenic for humans, has been tested in complex with several organic ligands, such as indole-3-butyric acid and indole-3-propionic acid.

Strikingly, proteasomal inhibition, as well as accumulation of ubiquitinated proteins and induction of apoptosis, were observed in MDA MB 231 breast cancer cells, whereas non-tumourigenic breast MCF10A cells were much less sensitive to the cadmium complexes, indicating cell-specific apoptotic death . Cadmium complexes with heterocycle-L-tryptophan Schiff base ligands such as 2-acetylpyrazine-L-tryptophan, 5-methylfurfural-L-tryptophan and 5-bromo-2thiophenecarbaldehyde-L-tryptophan have also been synthesized and tested for cancer specific proteasome inhibitory and apoptosis inducing activities. Results show that the inhibition of the proteasomal CT-L activity is strongly depending on the ligand; thus, while the Cd complex with 2acetylpyrazine-L-tryptophan and 5-methylfurfural-L-tryptophan were powerful inhibitors, the use of 5-bromo-2-thiophenecarbaldehyde-L-tryptophan as ligand produced an inactive complex (N. .

Manganese and gold complexes have also been investigated for their inhibitory activity on proteasome in cancer. In particular, the cefepime-Mn complex has been demonstrated to inhibit the proteasomal CT-L activity and to induce the apoptosis of breast cancer cells in a dose-and timedependent manner .

Gold(III) and gold(I) dithiocarbamate complexes have also been reported to be strong proteasomal CT-L activity inhibitors with IC 50 values ~ 1.1 μM. Interestingly, the different oxidation states of the gold ion seem to affect the mechanism of inhibition, as only the higher gold oxidation state has been reported to produce significant levels of reactive oxygen species (X. Zhang et al., 2010) .

Several additional gold complexes have been reported to have IC 50 in the μM range towards all three main proteasome catalytic activity, such as mononuclear gold, dinuclear(III) complexes and gold(I) phosphine complexes, whereas aurofin, an established gold(I) drug (currently in clinical use for the treatment of rheumatoid arthritis), has been demonstrated to be completely inactive in the modulation of proteasome activity (Micale et al., 2014) . The gold(III) complex AuL12

(dibromo[ethyl-N-(dithiocarboxy-kS,kS')-N-methylglycinate]) has shown attractive properties in terms of anticancer activity and toxicity. AuL12 was found to inhibit proteasome activity in living cells with an efficiency comparable to that of bortezomib (Tomasello et al., 2017) . Furthermore, AuL12 also inhibits Lys48-linked polyubiquitination in vitro at a concentration of about 7 µM, interfering with Ub activation reactions catalyzed by E1 enzymes. Another approach, based on the use of metal complexes to inhibit the function of the UPS system in cancerous cells, is to target deubiquitinases instead of the proteasome. As an example, nickel, as well as gold(I), pyrithione complexes were tested for their anticancer activity and it was found that both complexes are able to inhibit the UPS by targeting the 19S-associated deubiquitinases without directly affecting proteasome activity Zhao et al., 2016) .

Finally, it is worth mentioning that metal ions, such as copper, have a strong inhibitory effect also on other different enzymes (Grasso et al., 2011 (Grasso et al., , 2012 , which have been found to be associated with the proteasome and to be able to modulate its activity . For this reason, the use of metal complexes to modulate proteasome activity should consider all possible mechanisms and actors involved in the UPS system, including the interaction of the metal complexes with regulatory proteins, such as IDE .

Although proteins are generally found in the right folded and functional state in healthy cells, unfolded configurations are present, mostly occurring upon exposure to environmental stressors; furthermore, they may originate from multi-faceted alterations in translation, folding and intracellular trafficking. Under physiological conditions, the misfolded variant of proteins are J o u r n a l P r e -p r o o f either: (i) tagged for degradation via UPS or autophagy pathways, or (ii) correctly refolded back to the native state by chaperones, or else (iii) sequestered into intracellular compartments, such as aggresomes, which preserve them for the following refolding or degradation (Hartl et al., 2009; Chen et al., 2011; Escusa-Torret, 2013; Hipp et al., 2014; Sontag et al., 2014; Sweeney et al., 2017) .

Misfolded proteins can aggregate to form high-molecular weight species of different nature, such as soluble oligomers, prefibrillar species and highly ordered amyloid structure, which often consist of different aggregated-prone and normally folded proteins (Olzscha et al., 2011; Hong et al., 2011; Brettschneider et al., 2013; Sweeney et al., 2017; Olzscha et al., 2019) . Although there is compelling evidence that many proteins (if not all) can form amyloid-like structures under stressful conditions, nonetheless disease-associated amyloidogenic proteins are characterized by intrinsic structurally disordered elements in their free soluble form (Guijarro et al., 1998; Tzotzos et al., 2010) . Thus, a common hallmark of neurodegenerative diseases is the accumulation of misfolded protein aggregates into affected tissues, leading to a derangement of PN, and, ultimately, to progressive death of neurons (Bredesen et al., 2006; Goedert, et al., 2010; McAlary et al., 2019) . In Neurons, as well as each post-mitotic cell, are very susceptible to proteostasis unbalance mainly due to their long lifespan, morphology and enhanced metabolism (Tai and Schuman, 2008) . In particular, UPS is crucial in synaptic protein turnover, calcium flux dynamics, long-terminal plasticity and memory (Lopez-Salon et al., 2001; Colledge et al., 2003; Bingol and Schuman, 2004; Bingol, and Schuman, 2006; Fonseca et al, 2006; Guo, and Wang, 2007; Tai and Schuman, 2008; Djakovic et al., 2009; Wu et al., 2009; Djakovic et al., 2012) ; furthermore, besides the intracellular proteasome, a membrane-associated proteasome complex, specific for the nervous system, has been recently discovered (Ramachandran et al., 2017) . This complex seems to be involved in the modulation of neuronal function by degrading intracellular proteins into peptides that are then released into synaptic cleft, where they stimulate postsynaptic N-methyl-D-aspartate neuronal signalling (Ramachandran et al., 2017) . Impaired proteasome activity is reported in idiopathic neurodegenerative diseases, and some hereditary form of neurodegeneration is due to mutations in UPS components, such as PARK1 and PINK (see next paragraphs) (Ciechanover and Brundin, 2003; Ortega et al., 2007; McKinnon and Tabrizi, 2014; Thibaudeau and Smith, 2019) . A wide range of studies support the notion that a decrease of proteasome activity with age positively correlates with misfolded protein accumulation. This culminates then, in the presence of other J o u r n a l P r e -p r o o f pathological stressors at which aged people are exposed, with the progressive development of neurodegeneration Vigouroux et al., 2004; Mattson and Magnus, 2006; Zabel et al., 2010; Tomaru et al., 2012) . Accordingly, targeted proteasome inhibition in brain of animal models of neurodegeneration reproduces some clinical and neuropathological signatures of human diseases (McNaught et al., 2002 , McNaught et al., 2004 Ciechanover and Brundin, 2003; Bedford et al., 2008; Li et al., 2010) . Despite this evidence, it is still unclear whether reduced proteasome functionality is a primary event in neurodegeneration onset or the consequence of misfolded protein aggregation (Dantuma and Bott, 2014; Ciechanover and Brundin, 2003; Ortega et al., 2007; Thibaudeau and Smith, 2019) . Soluble oligomers, which are believed to be the most toxic and pathologically significant species among the different forms of aggregates, have been shown to impair proteasome activity (Cecarini et al, 2008; Tseng et al., 2008; Diaz-Hernandez et al., 2006; Bence et al., 2001; Deriziotis et al., 2011; Dantuma and Bott, 2014) .

Recently, a common mechanism has been proposed, according to which oligomers of different diseases-related proteins inhibit the 20S proteasome activity through an allosteric-driven interaction (Thibaudeau et al., 2018; . Specifically, the soluble oligomers, which adopt a similar three-dimensional conformation, were reported to bind the 20S, stabilizing the gate in the closed configuration. Oligomers-induced proteasome impairment seems then to be counteracted by HbYX peptides, which mimic the gate physiological opening induced by HbYX motifs of 19S ATPase (see also Section 2.3.2) (Thibaudeau et al., 2018) . Accordingly, α3Δn-HEK293 cells exhibit increased degradation of proteasome substrates, including neurodegenerative disease-related proteins (see also Section 2.3.2) (Choi et al., 2016) . These results support the scientific hypothesis that drugs which directly open the 20S gate, might have a relevant therapeutic potential in the management of neurodegenerations (Thibaudeau et al., 2018; Thibaudeau and Smith, 2019; VerPlank et al., 2019) . In this respect, it should be pointed out that the action of porphyrins (see Section 3.4.1) looks very promising (Di Dato et al., 2017) .

However, although it was generally accepted that, once formed, amyloid aggregates are resistant to proteasome degradation (Sweeney et al., 2017) , it has been recently reported that proteasome holoenzyme seems to possess a "fibril-fragmenting activity", being able to reduce the size of large tau and α-synuclein fibrils into smaller entities in vitro, thus opening a novel perspective in understanding proteasome role in neurodegeneration (Cliffe et al., 2019; Ye et al., 2020) . In the rest of this chapter, the contribution of proteasome to the onset and progression of main neurodegenerative diseases is reported. Additionally, we will focus on strategies developed so far to enhance proteasome activity.

J o u r n a l P r e -p r o o f

In the late 1901, the German neuropathologist Alois Alzheimer reported about the presence of amyloid plaques and neurofibrillary tangles (NFTs) in the brain of a woman suffering from progressive cognitive decline (Stelzma et al., 1995) . This was the very first paper reporting a case of senile dementia, a neurodegenerative disease that will be later commonly recognized as Alzheimer's Disease (AD). Almost 80 years after this ground-breaking report, protein aggregates present in amyloid senile plaques (i.e. amyloid peptides) (Glenner & Wong, 1984) and NFTs (i.e.

hyperphosphorylated tau) (V. M. Lee et al., 1991) were fully characterized. However, only over the last two decades, attention in the area of protein aggregation has increased considerably, transforming it into a key subject of study in diverse research areas ranging from chemistry to biology and medicine. The most important reason for the rising attention in this field is that most of the disorders are associated with amyloid aggregation (Chiti & Dobson, 2006) and neurodegeneration, which are becoming more and more expensive in terms of health care and social cost worldwide (Alzheimer's Disease International, 2010). After an initial enthusiasm in targeting amyloid protein aggregation as a possible therapeutic approach to treat AD, other protective mechanisms associated with properties of the cellular environment, such as the existence of molecular chaperones and degradation mechanisms have attracted increasing attention Morimoto, 2008) . Substantial shreds of evidence point to UPS malfunction as an important factor playing a key role in Aβ amyloid growth and AD pathogenesis. This is not surprising if one bears in mind that UPS surveillance is needed for a tightly regulated maintenance of all proteome components involved in memory formation, as well as synaptic plasticity and functioning (Lopez-Salon et al., 2001; Tai & Schuman, 2008; Djakovic et al., 2012) .

However, a deeper understanding of all components of the proteome quality control network is needed to allow us to envisage a successful regulation of all pathogenic pathways. As an example, it is critical to single out all the key components of the UPS, including the upstream processes, involved in AD pathogenesis to allow the design of small molecules with higher efficiency and less severe side effects (Cao et al., 2019) . In particular proteasome function (if compared to agematched controls) declines in AD brains, whereas other proteasome isoforms, such as immunoproteasome, are overexpressed in astrocytes (Keller et al., 2000; Nijholt et al., 2011) . As a matter of fact, several studies underscored that immunoproteasome is upregulated in glial cells surrounding Aβ plaques present in affected brains (Yeo et al., 2019) . The same work reported that YU102, a specific PI, abolished the production of inflammation cytokines from glial cells and J o u r n a l P r e -p r o o f improved cognitive performance in AD mice without any evident effect on Aβ plaques deposition.

Hence, the proteasome is now emerging as a major target in the treatment of memory loss and cognitive impairment in AD. (Hegde et al., 2019; Al Mamun et al., 2020) . A very recent study, involving 48 AD patients and 50 healthy volunteers, has clearly shown that proteasome levels are significantly decreased in erythrocytes of patients affected by AD. Moreover, the same study revealed that ubiquitin is overexpressed in red blood cells of AD individuals, thus suggesting that (i) the UPS is heavily involved in the pathogenesis of the disease and (ii) both Ub and proteasome may be investigated as AD biomarkers (Lv et al., 2020) . The role of proteasome inactivation in AD development, was addressed in vivo by studies using transgenic mice. In particular, APPswe/PS1de9 AD mice were crossed with mice expressing a green fluorescent protein (GFP) fused to a degradation signal (CL-1) targeted by the proteasome; these studies revealed that GFP protein-linked proteasome substrates build up in the hippocampus and cortex of AD mice at 4 weeks of age, and they were also confirmed by a concomitant accumulation of p53, an endogenous proteasome substrate, and of poly-ubiquitinated proteins. Altogether, these results suggest that the proteasome function is altered in AD mice even at a very young age, well before cognitive impairment and amyloid fibril deposition (Liu et al., 2014) .

Although it is known that Aβ is a proteasome substrate, it may also, in turn, inhibit 20S peptidase activity; in this respect, scanning transmission electron microscopy ( Hyperphosphorylated tau oligomers build up at synaptic and postsynaptic junctions in AD, and tau insoluble assemblies inhibit proteasome activity, leading to an accumulation of poly-ubiquitinated proteins (Myeku et al., 2016) . It has also been shown that CHIP (C-terminus of Hsp70-interacting proteins) E3 ligase, is overexpressed in AD patients and its downregulation brings out the accumulation of ubiquitinated tau proteins (Dickey et al., 2007; Petrucelli et al., 2004; Shimura et al., 2004) . Recently, it has been demonstrated that toxic Aβ oligomeric assemblies may bind proteasome, impairing its activity by interfering with gating phenomena. Moreover, as mentioned previously, other different proteins (e.g., α-synuclein and huntingtin), known to selfassemble into similar 3D structures, have the potential to inhibit 20S activity by a similar J o u r n a l P r e -p r o o f mechanism, thus pointing to a general oligomer-driven model of proteasome inhibition (Thibaudeau et al., 2018) . Notably, clearance of full-length monomeric tau was ATP-independent, whereas on the contrary, fibrillar tau hydrolysis was strictly related to the ATPase activity of the proteasome Besides Aβ/tau aggregation in toxic oligomers, other causes are known to come into play for proteasome impairment in AD, including abnormal generation of ROS (de Vrij et al., 2004) . Indeed, high intracellular levels of the redox-active metal ions Cu(II) are known to be associated to both Aβ and tau pathologies, even though it is known that only Cu(I) is internalized into the cells by the plasma transporter Ctr1 (Maryon et al., 2007) . In this respect, it should be outlined that Cu(II) ions are present at relatively high concentration in the brain and their levels are known to increase with age (Morita et al., 1994; Tarohda et al., 2004) . As a matter of fact, Cu(II) ions are also found in amyloid plaques present in the AD brain (Lovell et al., 1998; Suh et al., 2000) co-purifying with Aβ from AD brain tissues (Opazo et al., 2002) . Intriguingly, several Cu(II) complexes exhibit remarkable proteasome inhibitory capacities (Daniel et al., 2004; Marzano et al., 2009 ). All these findings reconcile with recent reports that demonstrate that Cu ( facilitating the ubiquitination of the β-secretase and its degradation by the proteasome, leading to a decrease of amyloid Aβ generation; notably, both Fbx2 and Uch-L1 increase synaptic plasticity and memory function in AD mice . It was also reported that UBB +1 , a ubiquitin form arising from a pathogenic mutation in the Ub gene through addiction of 19 residues to the Cterminus of the protein, inhibits proteasome activity and is present in neurofibrillary tangles of AD patients (Fischer et al., 2003) . Proteasome activation by exogenous agents may represent a promising strategy for AD therapy; in fact, feeding wild-type Caenorhabditis elegans with 18αglycyrrhetinic acid (18α-GA), a known proteasome activator, resulted in an increased proteasome activities and increased lifespan of worms. Moreover, higher proteasome activity was related to lower paralysis rates in C. elegans AD models. Notably, analogous promising results were confirmed also when murine and human neuronal cells were treated with 18α-GA (Papaevgeniou et al., 2016) .

A defective proteasome activity has been also related to increased levels of the APP-derived intracellular C-terminal membrane fragment β (CTFβ), a neurotoxic peptide with significant roles in AD pathogenesis (Bustamante et al., 2018).

Parkinson's Disease (PD) is a multi-factorial neurodegenerative disease, which primarily affects the nigro-striatal dopaminergic motor neurons. The pathognomonic histological lesion of PD, though not shared by all disease variants, is the formation of peri-nuclear intracellular aggregates, called Lewy Bodies (LB) (extremely rich in a proto-typical amyloidogenic protein, i.e. α-synuclein, and ubiquitinated proteins) through a process referred to as aggresome formation. It is still debated whether aggresome formation is a pathogenic event in PD progression or it rather represents a protective pathway activated to limit the high toxicity of the soluble amyloidogenic oligomers, which chronologically precedes aggregates formation (Wakabayashi et al., 2007; Wakabayashi et al., 2013; Raiss et al., 2016) . In support of this second hypothesis, which is the prevalent one, aggresomes formation is a phenomenon orchestrated by several proteins (including HSP10, p62, extensive crosstalk between the UPS and autophagy is expected to tightly balance aggresome growth and to avoid the protein imbalance of neurons . According to this PD pathogenesis framework, inherited and acquired conditions which predispose to the disease onset would impair this sophisticated mechanism of proteostasis maintenance.

Although very recently it has been reported that Ub-positive inclusions in LB mostly deal with K63 (e.g., autophagy-related) rather than UPS-specific K48 linkages, extensive studies on the genetically Furthermore, exceedingly high concentrations of metal ions, such as Zn 2+ , Cu 2+ and iron have been long detected in post-mortem brain biopsies (Le et al., 2014) ; in particular, in murine models it has J o u r n a l P r e -p r o o f been observed that, besides metal-related toxicity, Zn 2+ ions also trigger the expression and oligomerization-propensity of α-synuclein in nigrostriatal tissues and the selective loss of specific proteasome subunits, such as β5 and Rpt6 (Kumar et al., 2018) . Additionally, copper metabolism appears to be implicated in inducing cell death, since over-expression of α-synuclein and copper transporters stimulated loss of proteasome function, regardless of the tendency to form aggregates (Anandhan et al., 2015 Lan et al., 2016 .

A bulk of molecular evidences on PD pathogenesis comes from studies on genetic inheritance of the disease. Mutations of at least six genes have been linked with hereditary PD, namely α-synuclein (SNCA or PARK1), Parkin (PARK2), ubiquitin carboxyhydroxylase L1 (UCH-L1 or PARK5), PTEN-induced putative kinase 1 (PINK-1 or PARK6), DJ-1 (PARK7), and leucine-rich repeat kinase 2 (LRRK2 or PARK8) (Nuytemans et al., 2010; Janda et al., 2012) . Interestingly, all proteins, encoded by these genes, were found to interact at some level with proteasome or ubiquitinconjugating enzymes.

In particular, DJ-1 is an intracellular protein with pleiotropic activities which encompass cell morphology, functionality of mitochondria and ROS balance (Irrcher et al., 2010) ; most notably, it has been shown to positively regulate the transcriptional activity of Nrf-2 upon inactivation of PTEN and activation of PI3kinase/Akt/mTOR pathway. Nrf2 is a master regulator of anti-oxidant defence systems including transcription of proteasome genes (Niki et al., 2020) . A tight link between DJ-1 and Akt/mTOR pathway has been reported also in Drosophila melanogaster,

underscoring the degree of conservation across evolution of such a relevant pathway for neuron homeostasis (Yang et al., 2005) . Interestingly, detection of hyper-stimulated autophagy in DJ-1 deficient neurons is another indirect proof of the pivotal role of DJ-1 in stimulating the Akt/mTOR signalling, since this kinase is the major autophagy inhibitor. In this framework, it looks relevant to clarify the potentially controversial inhibitory activity of DJ-1 on the 20S catalytic activity both on synthetic and natural substrates, recently described (Moscovitz et al., 2015) . However, the authors propose that DJ-1 up-regulates the expression of proteasome subunits whilst repressing the catalytic activity of 20S assemblies, thus finely tuning the 20S proteasome bulk proteolytic activity. This activity would be necessary to address the cell need in balancing the clearance of oxidatively damaged proteins and that of native intrinsically unstructured proteins, which coordinate regulatory and signalling events (Moscovitz et al., 2015) .

Another gene, critically involved in PD, is LKKR2 and the clinical features of LRRK2-PD are often indistinguishable from idiopathic PD, with accumulation of α-synuclein and/or tau and/or ubiquitin in intraneuronal aggregates (Lichtenberg et al., 2011) . Although the mechanisms through which J o u r n a l P r e -p r o o f LKKR2 mediates toxicity are unknown, its mutation brings about a gain of functional mechanisms by means of an increased kinase-activity, which was shown to stimulate α-synuclein aggregation and cytotoxicity . Furthermore, LRRK2 overexpression in cells and in vivo downregulates UPS activity which turns out into the accumulation of intracellular substrates (Lichtenberg et al., 2011) .

Probably, the most studied protein in PD is the ring-finger E2-dependent E3 ubiquitin-protein Biological function of parkin deals with turn-over of a plethora of intracellular substrates.

A direct clue between parkin and α-synuclein was originally provided by identifying the selective ubiquitination of a specialised form of α-synuclein expressed in neurons, α22SYn (Shimura et al., 2001 ).

Additional substrates have been then identified, supporting a broader relevance of parkin activity in neuron homeostasis. A non-exhaustive list of substrates includes (i) CDCrel-1, which is the synaptic vesicle associated protein, (ii) p62, which is ubiquitinated at K13 by parkin, and (iii) p62 J o u r n a l P r e -p r o o f intracellular abundance, which follows an inverse linearity with parkin expression (Song et al., 2016; Okatsu et al., 2010) . Being p62 involved in aggresome formation, this finding elicits additional considerations on the cross-talk between the contribution of the key players of proteostasis in PD.

Another substrate of parkin is STEP6, which builds up in the striatum of PD subjects and also in murine models of the disease. The increase in STEP6, which follows parkin loss, is associated with a decrease in the phosphorylation of ERK1/2 and its downstream target, pCREB [phospho-CREB (cAMP response element-binding protein)] (Kurup et al., 2015) . Interestingly, dopamine signalling affects the de-phosphorylation of STEP6 and its catalytic activity; thus, in dopamine-deficient neurons STEP6 would be more active, depressing the ERK1/2 signalling pathway mentioned above, and grossly impacting on neuron homeostasis. Furthermore, STEP6 and BDNF are regulated through a reciprocal feedback mechanism and this may outline the loss of the neuron growth factor in PD.

Remarkably, one of the most relevant items about parkin biological role is its interaction with PINK, a Ser/Thr protein kinase encoded by the PINK1 gene, a major surveyor of mitochondria quality control, by targeting damaged mitochondria to autophagy-mediated clearance (e.g., In healthy mitochondria, PINK1 is quickly degraded by several mitochondrial peptidase, whereas in de-polarized mitochondria it is no longer cleaved and becomes exposed to the mitochondrial membrane, recruiting parkin for ubiquitination of various mitochondrial proteins thereby labelling the organelles for mitophagy (Greene et al., 2012) . Thus, down-regulation in parkin bioavailability turns out into a reduced clearance of damaged mitochondria which become a major source of ROS, dramatically contributing to the redox imbalance.

Furthermore, upon enzymatic shedding, PINK1 is further released as a soluble cytosolic form, called PINK1-s, which assists recruitment of parkin to the mitochondrial membrane and further contributes to the delivery of aggregating-prone protein to the forming aggresomes during proteasome inhibition. Thus, PINK1-s works as a sensor that links the proteasomal deficiency signal to the aggresome formation process Fedorowicz et al., 2014) .

As a whole, molecular insights on PD strongly support that tailored strategies of UPS modulation would provide a significant therapeutic efficacy in delaying disease progression.

Huntington disease (HD) is a neuro-degenerative disorder with autosomal dominant inheritance caused by a triplet expansion (i.e., CAG) in the huntingtin gene (Htt) which induces the protein to hold an exceedingly long poly-glutamine (polyQ) stretch at the N-terminus. A polyQ length >35 residues render pathogenic Htt (hereafter referred to as mHtt), which then acquires aggregatingprone properties (Di Figlia et al., 1997; Boland et al., 2018) ; the removal of the poly-Q stretch appears to be sufficient to rescue neuron homeostasis and to milder neuro-degeneration in murine models of HD (Zheng et al., 2010) . Abnormal synaptic transmission was reported to induce proteasome impairment in murine models of HD and a molecular mechanism through an increased cAMP signalling and the concomitant decreased PKA activity was proposed to explain this feature in neurons. Nonetheless, the cAMP/PKA pathway has been long considered central to HD pathogenesis by virtue of the pivotal role played by the two molecular pathways in neuron homeostasis and plasticity. The PKA holoenzyme, which is catalytically inactive in the absence of cAMP, is made up of two PKA-Rs and two catalytic subunits (Lin et al., 2013) ; in the presence of cAMP, cAMP-bound PKA-Rs dissociate from the catalytic subunits, which are then degraded by the proteasome. As a consequence, an impaired proteasome activity would allow the PKA-Rs to gather up reducing the amounts of free PKA catalytic subunits and thereby impairing PKA activity. On the other hand, PKA carries out the phosphorylation of Rpt6 at Ser120, a post-translational modification which enhances the activity of the proteasome and the constitution of capped particles (Lin et al., 2013) . Furthermore, during synaptic sprouting Rpt6 can also be phosphorylated by Ca 2+ /calmodulin-dependent protein kinase IIα (CaMKIIα) in a neuronal activity-dependent manner (Jarome et al., 2013) . Therefore, the HD onset might be correlated to a vicious cycle wherefore proteasome content drops down, due to its seizure, and the PKA-mediated stimulation of proteolysis fades out.

A significant contribution to HD pathogenesis further comes from the iPSC model of HD.

Remarkably, HD-iPSCs display constitutive increased proteasome activity, which was found to regulate the levels of both normal and mutated Htt, contributing to suppress polyQ-expanded Htt J o u r n a l P r e -p r o o f aggregation (Koyuncu et al., . As a matter of fact, HD iPSCs do not accumulate polyQ-expanded Htt aggregates even after multiple passages. Accordingly, a dysfunction in proteasome activity results in impaired Htt levels and aggregation of mHtt also in HD-iPSCs.

In iPSC, a major role in facilitating Htt clearance seems to be played by the E3 ligase UBR5.

Although loss of UBR5 did not impair pluripotency markers in human control iPSCs, it induced instead the formation of misfolded protein aggregates (i.e., aggresomes) (Koyuncu et al., 2018) .

The iPSCs experimental model has allowed to cast further light on the transcriptional activity of As mentioned above, several E3-ligases tag mutant Htt and are sequestered in aggregates. In different cell lines, mHtt clearance is usually carried out by canonical E3 ligases, such as UBR1, UBE3A, HSP and also non-canonical E3 ligases, such as Herp which, however, contain(s) a UBL domain (Luo et al., 2018) . Conversely, a role of de-ubiquitinase ataxin-3, involved in the pathogenesis of other neurodegenerative disorders (such as spino-cerebellar ataxia type 3), has not been confirmed in HTT progression by studies in murine models (Zeng et al., 2013) . Moreover, atypical ubiquitination of mHtt by some E3 ligases, such as WWP1, may favour disease progression .

Conversely, ubiquilin-1, a highly conserved family of proteins which facilitate protein disposal through autophagy and UPS and which is down-regulated in early HD, improves the clearance of Htt (Safren et al., 2014) Similarly, Usp14 has a favourable effect in cells expressing mutant Htt cells by decreasing the aggregate load and by enhancing cell viability (Hyrskyluoto et al., 2014) J o u r n a l P r e -p r o o f Finally, although UBE3A overexpression is known to promote UPS-mediated degradation of transfected mHtt in cultured cells, it is still unclear how UBE3A expression levels impact HD pathology. Remarkably, when the E3-ligase was up-regulated a drop in K63 ubiquitination of mHtt was observed (Bhat et al., 2014) . In this study, the presence of the pathological polyQ stretch was proposed to alter the overall folding of mHtt favouring the formation of K63 Ub linkages, also through cooperation of p62/SQSTM1, which are more prone to aggregation (Lim et al., 2015) .

Thus, to stimulate the UBE3A activity would be relevant to limit the toxicity of mHtt.

By virtue of its anatomical localization retina is also called the "window to the brain" ( (Naash et al., 1997; Fukuhara et al., 2001; Knowles et al., 2009; Fan et al., 2013) .

Moreover, surprisingly, murine transgenic models and human ex-vivo models of eye-diseases point toward a major role of non-canonical proteasome assemblies in retina development and J o u r n a l P r e -p r o o f homeostasis. A predominant contribution seems to be played by the immunoproteasome, by means of either PA28 expression and incorporation of inducible proteasome subunits in 20S assemblies, which is over-expressed to an exceptionally high degree in synaptic terminals and in photoreceptors (Hussong et al., 2010; Hussong et al., 2011; Shang et al., 2012; Lobanova et al., 2018; Aghdam and Mahmoudpour, 2016) . In detail, differentiation of murine retinal progenitor cells into their mature lineages requires the mTORC1-dependent STAT1 activation, which triggers the transcriptional upregulation of PSMB9 gene (which encodes for a catalytic subunit of immunoproteasome) but not of PSMB6 or PSMB7 (which encode for canonical subunits with trypsin-like and caspase-like proteolytic activities) (Choi et al., 2018) . Assembly of functional immunoproteasome is supposed to assist the 26S in clearing the bulk of short half-life proteins that accumulate in highly replicating cells (Choi et al., 2018) . Nonetheless, this finding envisages that the immunoproteasome might have a higher affinity than 26S for cell cycle substrates and for oxidized and unfolded proteins which may accumulate during replication. This last possibility is consistent with biochemical properties reported to date for the immunoproteasome and with the finding on a murine model of an inherited retinal degeneration wherefore photoreceptors carry a rhodopsin allele mutation (i.e., P23H); this mutation renders the protein unfolded and aggregating-prone, while the overexpression of PA28 counteracts the degeneration improving photoreceptor survival in such a murine model (Raule et al., 2014a; Raule et al., 2014b; Lobanova et al., 2018) .

The role of immunoproteasome and its substrate specificities gains further relevance when we consider that retina is an immune-privileged organ so that the processing of antigenic peptides, as well as their presentation, follow highly specific dynamics to regulate local immune response and immune-surveillance of this tissue, mostly concerning the maintenance of immune tolerance versus retinal self-antigens (Schuld et al., 2015; Voigt et al., 2017; Lipski et al., 2017; McPherson et al., 2014) .

Although indirectly, the Rett Syndrome (RTT) case (see also paragraph biogenesis) might further offer a clue for studying the specific regulation of proteasome biogenesis in retinal cells. RTT is a neurodevelopmental disorder classified as rare X-linked genetic disease (Amir et al., 1999) . In more than 95% of cases, girls, affected by the syndrome harbour a de-novo mutation in the MeCP2 gene, which encodes for an epigenetic transcriptional regulator with largely unknown biological functions (Chahrour et al., 2008) .

Whilst several brain areas display neuroanatomical abnormalities, retina as well as vision appear to be unaffected during RTT onset and progression (Jain et al., 2010; Rose et al., 2019) . underscoring onset and progression of these diseases are extensively discussed elsewhere and will not be discussed herein (Tarr et al., 2013; Lombardo et al., 2013; Parravano et al., 2013; Picconi et al., 2018) . However, they represent two eye disorders wherefore UPS and alteration of the PN appear to follow a specular pattern and for which pharmacological strategies targeting the UPS might provide a valid therapeutic opportunity.

Contribution of the UPS in DR onset is poorly studied, even though several lines of research support a pivotal role played by the proteasome in regulating the nuclear activity of key transcription factors and the release of cytokines in the early vascular response to the hyperglycemic insult (Aghdam et al., 2013; Campello et al., 2013; Rahimi et al., 2012) .

Although choroidal endothelial cells seem to contribute only marginally to this phenomenon, an aberrant proliferation of retinal endothelial cells (which ultimately lead to micro-haemorragic lesion, vessel leakage and irreversible fibrosis) follows the increased secretion and bio-availability of VEGFs. Several independent research teams suggest that the retinal cell type, which first sense the hyper-glycemia is the Muller glia, and indeed metabolism of this cell type appears to be sensitive, through unknown mechanisms, to fluctuations in glucose concentration, as those occurring in vivo in diabetic subjects Le,2017; Picconi et al., 2019; Picconi et al., 2017; Voigt et al., 2017; Matteucci et al., 2014) . This insult stimulates the secretion of VEGFs as well as of other pro-inflammatory cytokines. VEGFs synthesis is mostly regulated by the transcriptional activity of NF-kB and HIF-1α whose nuclear translocation is regulated by J o u r n a l P r e -p r o o f proteasome proteolytic activity (see Section 3.2.1) (Alkalay et al., 1995; Traencker et al., 1994; Chen et al., 1995; Ferrara et al., 2004) .

Therefore, a better understanding of the molecular mechanisms of enhanced protein turn-over upon hyper-glycemia in Muller glia and, possibly, in additional retina cell types would allow to envisage selective therapeutic approaches to target VEGF synthesis rather than its biological cascade once secreted. In fact, clinical regimens for DR treatment mostly deal with VEGF inhibitors which however do not distinguish between the physiological and pathological angiogenesis with the former being as much relevant for retina homeostasis as blocking the latter would be for DR progression (Ferrara et al., 2004; Lacal et al., 2018) . Thus, selective targeting of factors, that regulate the proteasome-mediated turn-over of players in DR progression, would help to overcome the limitation of traditional proteasome inhibition strategies, which stop bulky proteolytic burden thereby compromising PN.

The involvement of proteasome in the onset of glaucoma appears to follow an opposite path to that observed in Muller glia cells in DR. In fact, there is a compelling evidence that PN might be dysregulated in both main clinical forms of glaucoma, namely (i) primary open-angle glaucoma (POAG) and (ii) normal-tension glaucoma Quaranta et al., 2016; Agarwal et al., 2009; Weinreb et al., 2014; Wunderlich et al., 2002; Caballero et al., 2003; Caballero et al., 2013) .

In this regard, the term glaucoma encompasses a heterogeneous group of neurodegenerative disorders characterized by the loss of retinal ganglion cells (RGC) and atrophy of the optic nerve their axons generate Swarup and Sayyad, 2018; Minegishi et al., 2016) . leading to RGCs loss, optic nerve degeneration and visual decline. Therapy with glucocorticoids has been long known to induce acute iatrogenic form of glaucoma by affecting the TMC metabolism, but the primary alteration of such a cell after GC administration is unknown (Roberti et al., 2020) . Interestingly, TMCs express a ubiquitous protein, called myocilin (i.e., myoc) (i.e., from the MYOC gene), whose expression is up-regulated in TMCs when exposed to GC, but also oxidative stress and cytokines (Qiu et al., 2014; Wang et al., 2019; Resch and Fautsch et al., 2009; Micera et al., 2016) ; when over-expressed, this protein is supposed to pose a metabolic threat to

TMCs through unexplored gain of function mechanisms Jain et al., 2017) . This pathogenic effect may occur either in the intracellular or extracellular compartments, in accordance with the broad localization of the protein. Furthermore, mutations in MYOC gene are the most studied cause of the juvenile form of glaucoma and, among the different point mutations described so far, the most prevalent ones (e.g., Pro370Leu) render myocilin amyloidogenic and aggregatingprone (Yam et al., 2007; Wang et al., 2019) . Myocilinis a proteasome substrate, and, in the absence of GC therapy, proteasome activity appears to decline in TMCs culture isolated from patients suffering from glaucoma in an age-dependent manner; further, myocilin expression in HeLA cells was found to decrease the bioavailability of some 20S subunits (Qiu et al., 2014) . Conversely, the GC effect on MYOC processing and proteasome regulation is unknown. Nonetheless, the role of proteasome impairment in driving cell senescence and the role of proteasome re-activation in delaying this phenomenon is well studied in several cell types but not in TMCs Deschenes-Simard et al., 2014) .

Even though transgenic models do not always support an unequivocal role of MYOC in glaucoma onset, the interactome of this protein is worth being studied to address the metabolism of TMC Senatorov et al., 2006; Zhou et al., 2008; Joe et al., 2015; Jain et al., 2017) .

Therefore, to study the dynamics of myocilin digestion in TMCs and how proteasome might undergo dysregulation under metabolic conditions that are commonly seen in glaucoma subjects, it might help to explore the molecular insights of pharmacological strategies based on UPS rescue.

Differently from POAG, normal-tension glaucoma is not supported by an increased intra-ocular pressure and the degeneration of the optic nerve likely depends on a primary insult on RGCs. Even in this case, a tight involvement of intracellular proteolytic pathways is largely envisaged.

Optineurin (OPN) gene encodes for a protein involved in intracellular vesicle trafficking, and expression of mutated forms of optineurin induces a severe dysregulation of the UPS and of autophagy Shen et al., 2011; Sirohi et al., 2016) .

Besides the molecular findings discussed above, a major suggestion for the relevance of proteasome in handling the PN in retina comes indirectly from biochemical pharmacology (Sbardella et al., 2020b) . Several clinical trials worldwide support the therapeutic efficacy of citicoline for glaucoma treatment (Roberti et al., 2015; Parisi et al., 2008; Parisi et al., 2018; Parisi et al., 2019; Carnevale et al., 2019) . Citicoline, also known as CDP choline, is a drug made up by choline and cytidine diphosphate which displays optimal bioavailability and easily crosses the blood brain barrier (Faiq et al., 2019) . Although the mechanisms of action of citicoline have never been identified at molecular detail, its wide usage in clinical regimens is based on its outstanding safety profile and on the efficacy also in neurological disorders, including neurodegenerative disease, such as the early phases of AD and PD onset, though the trials having been run are still limited (Eberhardt et al., 1990) .

Our group has very recently reported that citicoline is an allosteric modulator of proteasome in vitro and in vivo, wherefore citicoline binds the 20S with a very high affinity (i.e., in the low nanomolar range), stimulating the clearance of synthetic substrates as well as of α-synuclein (Sbardella et al., 2020b) . Surprisingly, in neuron-derived cells, citicoline was found to both stimulate 20S activity and to promote the assembly of proteolytic active capped assemblies (i.e., 26S and 30S, see Section 2.3.2) (Sbardella et al., 2020b) . As a matter of fact, cells stimulated with citicoline experience a very significant increase in the overall proteolytic burden by the UPS. Therefore, although it is reasonable that the proteasome stimulation is not the only therapeutic effect, the neuro-protective role of citicoline highlights the relevance of proteasome functionality in maintaining the postmitotic cells homeostasis. Nonetheless, citicoline experience in clinical trials might be looked as a proof of concept that the activation of UPS is a valid strategy to delay the progression of pathologies sustained by proteo-toxicity.

Neurodegenerative diseases are clinically heterogeneous proteinopathies sustained by the accumulation of aggregates of misfolded disease associated proteins (see also Section 4.1). The prevalence and incidence of neurodegeneration increases dramatically with age, and, since people life expectancy rises worldwide, also the number of individuals suffering from these pathologies is expected to dramatically increase in the next years (McAlary et al., 2019; Jones and Tepe, 2019) .

Despite social and economic relevance, no effective cure still exists; therefore, the development of novel therapeutic approaches is essential.

As discussed above (see Section 4.1), UPS alteration contributes to disease onset and progression, leading to an intense research effort with the purpose of identifying therapeutic strategies targeting UPS. Different approaches to enhance UPS functionality have been proposed, spanning from stimulation of ubiquitination and/or inhibition of de-ubiquitination (Box 2), or inhibition of protein aggregation, being this last strategy founded on evidences that monomeric proteins are better degraded by proteasome than oligomers (Dantuma and Bott, 2014; Wertz and Murray, 2019) . In addition, modern strategies envisage the direct proteasome stimulation by either (i) drugs which specifically target proteasome particles increasing their bulk proteolytic activities (see also Section 2.2.1) or (ii) phosphorylation of proteasome subunits (Myeku and Duff, 2018) .

According to the first point, the identification of "drug-like" molecules, which directly activate proteasome, is challenging. Notably, a chemical genetics screening of over 2750 compounds using a proteasome activity probe as a readout in a high-throughput live-cell fluorescence-activated cell sorting-based assay has led to the identification of more than ten compounds that increase proteasome activity (Leestemaker et al., 2017) . A promising, but still poorly explored strategy, is the development of therapeutic peptides and/or peptidomimetics, designed on the basis of specific binding regions of natural proteasome regulators (Wilk et al., 1997; Fosgerau et al., 2015; Jones and Tepe, 2019) . In general, the advantage of peptide usage seems to deal with the higher specificity and selectivity with respect to molecular target; however, beside the complex synthesis, they suffer from low metabolic stability and poor membrane permeability (Fosgerau et al., 2015; . The most common class of synthetic peptide acting as proteasome activators are based on the HbYX motif (see Sections 2.3.2 and 4) . In this context, it has been shown that peptides derived from C-termini Rpt2 and Rp5, and a 14-mer peptide, based on the Cterminal fragment of Blm10 (the yeast ortholog of PA200, see box 1) and containing the HbYX motif, efficiently stimulated proteasome activity in vitro (Sadre-Bazzaz et al., 2010; Karpowicz et al., 2015) . Recently, it has been reported that upon introduction of the HbYX sequence, the proline-and arginine-rich peptide (PR11), which is a 20S allosteric inhibitor, turned to be a proteasome activator in vitro and in cell model (Osmulski et al., 2020; . Another example of a "drug-like" molecule with a different unknown mechanism of action is the proteasome-activating peptide 1 (PAP1), which increases the chymotrypsin-like proteasomal catalytic activity in vitro and in cell models and is further able to halt protein aggregation (Dal Vecchio et al., 2014) . Among proteasome activators, natural compounds, such as oleuropein, betulinic acid and fatty acids deserve particular attention, and their features will be discussed in the next sections.

Concerning the phosphorylation strategy, a bulk of studies have shown that reversible phosphorylation of proteasome subunits positively regulates its function (VerPlank and Goldberg, 2017; Myeku and Duff, 2018; VerPlank et al., 2019) . Accordingly, protein kinase A (PKA)-dependent phosphorylation is involved in the regulation of multiple aspects of proteasome functionality, such as: (i) enhancement of Rpt6 ATPase activity through phosphorylation which further stimulates the association of 20S and 19S in vitro (Box 3); increase in the proteasome capacity to clear ubiquitinated proteins, peptides and ATP as well as the degradation of aggregation-prone proteins in cells upon phosphorylation of Rpn6 at serine14 (Box 3) Lu et al., 2008; Jarome et al., 2013; Asai et al., 2009; Lokireddy et al., 2015) .

Moreover, phosphorylation of Rpt6 by kinase CaMKIIα induces 26S translocation into dendritic spines in primary neurons, promoting local protein breakdown and driving the formation of synaptic connections (Djakovic et al., 2009; Bingol et al., 2010; Hamilton et al., 2012; Jarome et al., 2013) .

Therefore, proteasome subunit phosphorylation has been suggested to rescue proteasome function and it could represent a promising strategy to treat neurodegeneration (VerPlank and Goldberg, 2017; Myeku and Duff, 2018) . Recently, it has been reported that, in hyppocampal neurons, only 20% of proteasome seems to be in a "active" substrate-engaged state, whereas the remaining part is in an "inactive" substrate-accepting ground state. Therefore, it has been speculated that phosphorylation increases the percentage of active forms of proteasome, recruiting "idle" particles as well as directly stimulating their activities (Asano et al., 2015; Myeku and Duff, 2018) . As a matter of the fact, a promising strategy should be the stimulation of PKA activity through the modulation of the amplitude of cAMP signal. The cAMP level is curtailed by cyclic nucleotide phosphor-di-esterases (PDE), which acts negatively by regulating PKA signals. Inhibition of PDE stimulates cAMP/PKA axis and activates proteasome, opening to a novel potential use of PDE inhibitors in the CNS diseases treatment (VerPlank and Goldberg, 2017; Myeku and Duff, 2018) .

Accordingly, it has been shown that PDE4 selective inhibition by rolipram induces phosphorylation of several subunits of 26S, leading to an increase in mouse models of UPS-mediated clearance of tau and amyloid aggregate, accompanied by a reduced cognitive impairment (Vitolo et al., 2002; Smith et al., 2009; Myeku et al., 2016) . Moreover, cAMP/PKA axis activation, which follows PDE10 inhibition, reduces Htt aggregation through a proteasome-dependent mechanism, and ameliorates motor and cognitive deficit in Htt mouse model (Giampà et al., 2010; Lin et al., 2013; Beaumont et al., 2016; Harada et al., 2017) . Furthermore, the administration of the FDA approved PDE3 inhibitor, cilostazol, to a mouse model of tauopathy enhanced proteasome function and attenuated the tauopathy and cognitive decline in rTg4510 mice, suggesting that this drug could be potentially repurposed for the treatment of patients with early-stage tauopathy (Schaler and Myeku, J o u r n a l P r e -p r o o f 2018) . As a whole, despite some early encouraging results in mouse model, it seems clear that the broad range of biological functions, mediated by cAMP, can reduce the clinical efficacy of PDE inhibitors, due to their adverse effects (Myeku and Duff, 2018; Heckman et al., 2018) . It is worth recalling that cAMP/PKA pathway transduces the intracellular signalling of a number of hormones:

thus, in such a way, proteasome function can be regulated by hormonal and metabolic stimuli (Box 3) (VerPlank and .

Many organisms have developed a large number of small molecules, which modulate the activity of UPS components (Rousseau and Bertolotti, 2016) . Those of nutraceutical origin, (contained in fruits, in vegetables and their extracts) are very attractive for their positive effects as antiaging and in the treatment and the prevention of a wide range of pathologies. In fact, dietary phytochemicals exhibit broad and different biological activities, including antioxidant action, free radical scavenging, anti-inflammatory and metal-chelating properties, that represent the evolutive result of the vegetable system defence. All of them are secondary metabolites that plants produce to counteract against various stresses (Murakami, 2013) ; thus, they can be considered "multifunctional drug-like molecules", and it is not surprising such high variety of targets, since they are small molecules with very simple chemical structures.

Concerning the activity of these compounds on proteasome, those able to activate/enhance proteasome activity are rare, (Bonfili et al., 2008; Dahlmann, 1993; Huang and Chen, 2009 ) and they are often characterized by an ambivalent behaviour, acting alternatively as inhibitors and/or activators according to diverse conditions. As an example, the effects of curcumin (Cuanalo-Contreras and Moreno-Gonzalez, 2019) (1E,6E)-1,7-bis-(4-idrossi-3-metossifenil)-epta-1,6-dien-3,5-dione) on the UPS reflects the hormesis principle (e.g., the biphasic dose-response to an environmental agent characterized by a low dose stimulation or beneficial effect and a high dose inhibitory or toxic effect), being characterized by an inverted U shape dose-response (Ali and Rattan, 2006) ; thus, curcumin treatment (up 1 µM for 24h) increases proteasome activity in keratinocytes, but it displays an inhibitory effect at 10 µM (Murakami, 2013) . In particular, curcumin induces 26S perturbation, leading to an impairment of cell proliferation in various cancer cells and reduction of cancer burden in mice (Banerjeeaet al.,2018) .

By analogy, quercetin (3,3',4',5,7-pentahydroxyflavonethe), the most abundant flavonoid found in fruits and vegetables, which has been initially reported to be a 20S inhibitor (Chen, 2005) (IC 50 = J o u r n a l P r e -p r o o f 3.5 μM), it has been shown to enhance the proteasome activity in vivo and to reduce the Aβinduced toxicity in a dose-dependent manner when administered to a Caenorhabditis elegans AD model (Chondroianni et al., 2010) . Likewise, the polyphenol resveratrol, that was previously described as a natural direct PI Qureshi et al.,2012) , recently has been reported to enhance proteasome activity recovering the impaired proteostasis in a C. elegans AD model, and in AD transgenic mice (Regitz et al., 2016) ; in addition, resveratrol has been shown to enhance cognitive activity by increasing 20S proteasome subunits levels and stimulating proteasome activity (Corpas et al., 2019) .

Hereafter, we focus only on bioactive compounds that directly target the naked catalytic particle, 20S, thus enhancing the ubiquitin-ATP-independent proteolysis, the main pathway degrading the oxidatively damaged and intrinsically disordered proteins (Ben-Nissan and Sharon, 2014) .

Oleuropein, the most abundant phenolic compound extracted from Olea europaea (leaf and olives), enhances all three proteasome activities in vitro and promotes cellular resistance to oxidants, prolonging human fibroblasts lifespan (Katsiki et al., 2007) . Furthermore, it indirectly promoted the enhancement of proteasomes activity by regulating rpn1 expression and downregulation of the apoptosis pathway gene, egl-1 (Tsai, et al., 2017) . Interestingly, betulinic acid has shown a neuroprotective effect in vascular dementia rat models, re-establishing the cerebral blood flow, restoring behaviour parameters and significantly improving the BDNF levels, with a restrain of the oxidative stress and of inflammatory parameters (Kaundala et al., 2018) .

Besides the proteasomal effect, oleuropein and betulinic acid could be also considered as pleiotropic small molecules, for their anti-HIV (Mayaux JF et al., 1994; Yang et al., 2016) and anti-tumour activity towards some cancer cell lines (Kessler et al.,2007; Pisha et al., 1995; Saeed et al., 2018) .

Although both oleuropein and betulinic acid have been previously reported to be natural proteasome activators (Darci et al., 2017) , some authors recently clarified that the stimulatory activity is J o u r n a l P r e -p r o o f restricted to fluorogenic substrates, and no effect has been reported on the turnover of a mis-folded protein in vitro or in living cells.

Among naturally occurring activators of 20S proteasome there are some physiological cellular components, such as mucopolisaccarides (e.g., heparin), glycolipids (e.g., ceramides, lysophosphatidyl-inositol and cardiolipin) (Ohkubo et al., 1991; Matsumura and Aketa 1991; Ruiz de Mena, 1993) , and some proteins (not included in the physiological UPS control), such as the arginine-rich histone H3, a chromatin binding protein able to selectively enhance protein degradation by the proteasome (Orlowski, 2001) .

The detailed mechanism by which these compounds regulate 20S degradation is still largely undetermined, but it might be related to the gate opening, mimicking the RP (or naturals Proteasome Activators) interaction (see Sections 2.2.1 and 2.3.1).

Fatty acids are likely the first small molecules that were first described as 20S proteasome modulators. Indeed, our basic knowledge of proteasome function is rooted on the pioneering studies conducted throughout the 80s and 90s (Orlowski and Wilk ,1981; Ishiura, 1986; Folco, 1988) , using fatty acids and SDS as activators. The first systematic study on proteasome peptidase activity reported the effects of several saturated and unsaturated fatty acids examining various carbon-chain lengths and underlining that the optimal 20S activation potency may be achieved by using fatty acids with a C18-C20 chain carbon; thus, the oleic acid resulted the most active compound, with an activating effect 50-fold higher than SDS (Dahlmann et al., 1985) . Although our knowledge of proteasome structure and function was still in its infancy, in those early reports it was proposed that activation mechanism could be basically related to conformational changes occurring in the enzyme. Furthermore, they also proposed that fatty acids, abundant in muscle, could participate in the physiological regulation of proteasome-mediated protein degradation. Later on, Orlowski et al.

performed a detailed kinetic analysis, reporting that lauric acid activates the PGPH activity like SDS; increasing concentrations of lauric acid caused a shift in the apparent K m toward lower substrate concentrations with a concomitant increase in V max (Orlowski et al. 1991) . However, unlike the SDS-mediated one, this activation occurred with a sigmoidal shape of the velocity curve,

suggesting the presence of two (or more) substrate binding sites interacting cooperatively. In other words, in the absence of an external activator only part of this activity is manifested, thus underlining the cooperative control of allosteric sites and the concepts of "latency" and of "multi-J o u r n a l P r e -p r o o f proteasic complex"; however, there is still some controversy concerning the ability of most fatty acids to enhance the three main 20S proteolytic activities. Actually, fatty acids often exhibit a double-faced nature, behaving as activators and/or inhibitors, according to the type of activity measured. In ostrich liver the PGPH proteasome activity was found to be activated by all (with the exception of decanoic acid) types of fatty acids in a concentration-dependent fashion, whereas the chymotryptic and tryptic-like activities were differentially inhibited (Klinkradt et al., 1997 ). An attempt to clarify the intricate mechanism by which the three peptidasic activities of 20S proteasome are regulated by fatty acids was made by Yamada and co-authors, who reported that the pattern of activation of the trypsin-type peptidase is distinctly different from those of ChT-L and PHPH-L; thus, linoleic and oleic acids strongly activated both the chymotrypsin-type and the peptidyl glutamyl-peptide (PGP) hydrolase-type activities in a biphasic activation pattern (Yamada et al.,1998) . Conversely, the activation pattern of tryptic-type peptidase occurs in a tri-phasic manner through an inhibition over the low concentration range, activation in a middle concentration range and inhibition again over a higher concentration range. These apparently conflicting results were explained by hypothesizing the existence of two classes of binding sites, namely "latency sites" and "activation sites", and the fatty acid activation or inhibition phenomena have been interpreted as the result of binding to these different sites.

Over last decades, the studies on isolated/purified proteasome have been replaced by investigations performed through cellular or clinical studies. Some of these reports focused on the role of polyunsaturated fatty acid on protein-breakdown in muscle mass of cachectic cancer patients. As an example, the neuroprotective effects of (Undurti et al.

Acids (LCPUFA) have been ascribed to some modulatory effects on the UPS, albeit no evidences on direct interaction with 20S proteasome have been reported. Docosahexaenoic acid, the most unsaturated omega-3 fatty acid, displaying pro-apoptotic activity against tumour cells, was reported to exert its anti-cancer activity acting on UPS, even though no evidence of a direct interaction with 20S has been reported (Jing et al., 2014) . There is also evidence describing the inhibitory (Hamel, 2009 ) effect on proteasome by the saturated fatty acid palmitate; this fatty acid is believed to contribute to type-2 diabetes, blocking UPS activity with a consequent lipotoxic effect on pancreatic Furthermore, a greatly diminished capacity to stimulate the 20S was observed when the phenolic amide was substituted by aryl groups. These derivatives still need a careful pharmacological evaluation, also taking into account that, beyond their role as a nutritional energy source, fatty acids have several molecular targets, such as enzymes, receptors, and they are increasingly considered as important signalling molecules that can induce several physiological and pathophysiological effects.

Although over the last decade our knowledge of human diseases has greatly increased, its translation into new drugs and therapeutic benefits has been much slower than expected (Ashburn et. al.2004; Scannell et al., 2012) The reasons that may explain this apparent incongruity are multifaceted and include the increased time needed to pipeline new drugs to the market, a high attrition rate of drug candidates in clinical trials (Pammolli, 2011) , and rapidly changing regulatory requirements. Some reports estimate that, on average, for every dollar invested by the pharmaceutical industry in research and development (R&D) less than a dollar is returned, thus suggesting that investments on R&D will rapidly decline in the very next future (Pushpakom et al.,2019) .

Therefore, drug repositioning (or repurposing), an approach to identify new medical applications for drugs, already approved for different therapeutic uses, (Nosengo, 2016) offers a number of advantages over the development of entirely new drugs, such as (i) the reduced costs in the case of failure, and (ii) the shorter time interval for the transfer to the market because safety assessment has been already completed. Thus, given the urgent need to find a treatment for neurodegenerative diseases (such as AD, PD and ALS), it is not surprising that an increasingly large number of existing drugs are tested for these disorders; in this respect, an important example is the repositioning of galantamine, one of the drugs now available on the market for the treatment of AD (Durães et al., 2018) .

Despite many examples of drug repositioning have been based on serendipity, a rational development of a repurposed drug implies a detailed knowledge of the pathways involved. As an example, there is evidence that AD development is associated to aggregation of the neurotoxic amyloid β (Aβ) peptide (Kang, 1987) , being the consequence of a failure of proteasome function and a consequent accumulation of poly-ubiquitinated substrates, as detected in AD neuronal tissues (Perry, 1987) . In particular, in response to the increased oxidative and proteotoxic stress, the J o u r n a l P r e -p r o o f percentage of uncapped 20S proteasome is significantly increased in AD neuronal tissues in comparison to healthy cells . Moreover, the Aβ peptide, as well as other intrinsically disordered proteins (IDPs), is a substrate of the 20S proteasome.

On these premises, it has been reported that pyrazolones, a class of small molecules extensively used in the past as painkillers and antipyretic drugs, induce proteasome activation in mice models of ALS (Trippier, 2014) . In a recent report, it was demonstrated that some members of this class of molecules (i.e., aminopyrine, 4-aminoantiypirine and nifenazone) may enhance ChT-L proteasome activity in tube tests (Santoro et al., 2019) . Proteasome activity assays, carried out in parallel in the presence of an excess of reducing agents (i.e., ascorbic acid or glutathione), underscored that proteasome activation by pyrazolones is not directly related to their antioxidant properties, thus suggesting that an alternative mechanism of action should be proposed. In the case of aminopyrine the evidence that it is able to activate native 20S, but it is ineffective on a mutant (i.e., α3ΔN, which has a permanently opened gate, see Section 2.2.2), envisaged that its effect on proteasome activity is mainly related to enhanced dynamics of the outer α-rings, which is a common mechanism of proteasome activation by small molecules (see Section 2.2.2) (Njomen, 2019). Furthermore, the effects on the different cellular proteasome forms (i.e., uncapped 20S, singly capped 26S and doubly capped 30S, see Sections 2.2.2 and 2.3.2) were also assayed in neuroblastoma SH-SY5Y cells by separating proteasome assemblies in non-denaturing gels. It was observed that proteasome assemblies resulted significantly stimulated two hours after the treatment with the drug, even though this effect vanishes over 24 h after stimulation, reflecting the pharmacokinetics properties of aminopyrine (with a half-life in blood serum of approximately 2 h). Docking simulations, performed using aminopyrine, antipyrine, 4-aminoantipyrine and nifenazone as ligands of human 20S, have outlined that they interact with α-rings, involving the α1/α2 and α5/α6 binding pockets; the most active molecules display a binding free energy ~ 30 Kcal/mol more favorable than the less active ones. In particular, it was observed that aminopyrine bridges α1and α2 subunits since its phenyl ring is involved in hydrophobic interactions with residues L22, Y25, E26, A126, and A157 of the α1 subunit; the oxygen atom of aminopyrine is also H-bonded with Y159 and the N-methyl group with residue A32 of the same subunit. Moreover, residues G30, G31 and A32 of the α2 subunit are linked by non-polar interactions to the 4-(dimethylamino) group of aminopyrine. Next, T-shaped stacking interactions bridge the residue F162 of the α5 subunit with the phenyl ring of aminopyrine, and the residue Q60 of the α6 subunit turns out to be H-bonded to the oxygen atom of the ligand. It is important to remind here that the α1/α2 grooves are the preferential anchoring sites of the HbYX motif which binds the 20S with the Rpt3 subunit of the regulatory particle 19S (see Sections 2.2.2 and 2.3.2) ; furthermore, small molecules, known to mobilize 20S J o u r n a l P r e -p r o o f gating dynamics, bind the α5/α6 grooves (Di Dato et al., 2017) . Notably, the inactive compound antipyrine mainly interacts through T-shaped aromatic interactions and H-bonds with two residues of α2 subunit Y159 and Y160, respectively, but no bridging interactions with any other subunit of the α-ring are observed ( Figure 5 Aβ proteotoxicity with a mechanism of protection mostly related to proteasome activation. These results are likely to stimulate further studies focusing on proteasome activation by repurposed drugs and, ultimately, to relaunch investments from pharmaceutical industries in this risky area.

Although not deeply investigated yet, proteasome inhibitors discussed so far display a known antiinflammatory activity, envisaging a therapeutic efficacy in combined regimen in subjects with acute severe inflammatory processes, such as in viral infections. This potentiality assumes a particularly updated importance nowadays during the recent pandemia due to SARS-Cov-2, which is likely to infect millions of people worldwide with a significant lethality rate. Besides the public health and victim tolls, which are by far the most urgent topics, disease spread is dramatically impacting on world social and economic activities, making even more urgent the identification of a specific therapy or a vaccine (Andersen et al., 2020; Sheeren et al., 2020; Baden and Rubin, 2020; Lipsitch et al., 2020) .

The limited clinical and laboratory data available so far suggest that in most cases SARS-Cov-2 infection evolves through symptoms overlapping those of canonical flu, even though a very large number of subjects do not develop symptoms (Baden and Rubin, 2020; . In a limited, but significant, especially for the health assistance burden, number of cases, infection progresses toward a clinical picture of interstitial pneumonia sustained by the massive stimulation of the immune system the virus appears to be able to elicit (Baden and Rubin, 2020; .

The cytokine storm which underscores this disease progression often induces Acute Respiratory Distress Syndrome (ARDS) and diffuse thrombotic angiopathy which are, to date, the prevalent cause of death of ill patients.

Nonetheless, the massive inflammatory response rather than virus replication is gaining increasingly relevance as the real target of therapy. This is emphasized by the apparent efficacy of therapies based on biological drugs which target the pro-inflammatory cytokines (Baden and Rubin, 2020) . In any case, specific trials will definitively address the therapeutic efficacy of these approaches, hopefully allowing to identify a therapeutic regimen which can be early undertaken to prevent disease complication and sanitarian costs. including the marked innate immune inflammatory cytokine release (Ma et al., 2010) .

Among studies, reported to date, there is a compelling evidence that the UPS could regulate the virus infectious cycle at multiple levels, with the exception of virus internalisation which occurs through endocytosis following recognition of Angiotensin Converting Enzyme-2 (ACE2) receptor on host cells by the spike protein (S) (Mathewson et al., 2008; Li et al., 2007; Li et al., 2003) .

Interestingly, chemical inhibitors of proteasome induced the virus particles to accumulate in late endosomes and lysosomes, suggesting a UPS role in virus release from endosomes (Yu and Lai, 2005) . Once released in the cytoplasm, the nucleic acid of SARS-Cov (a positively single stranded RNA) encodes four structural proteins, nucleocapsid (N), envelope (E), membrane (M) and spike (S) proteins and about 16 non-structural proteins which are translated as a single poly-protein (Yu and Lai, 2005; Li et al., 2007; Schneider et al., 2012) . The highly antigenic protein N of SARS-CoV, which is an extensively glycosylated and positively charged protein of the nucleocapsid, forms a helical ribonucleoprotein complex with the viral RNA, and it was found to interact with ATPase 6 (i.e., a 19S subunit) in lung fibroblasts infected with the virus .

Therefore, although further studies are required to confirm this hypothesis, a direct down-regulation of proteasome complexes by SARS-CoV can be envisaged, since in the intracellular space the ATPase 6 subunit (because of its position in the multi-subunits complex, see Studies on non-structural proteins, which intervene either in the virus replication and in the interaction with the host machineries, besides confirming that the UPS does not affect virus internalization, envisaged that virus-UPS interaction may account for the mechanisms of immune system evasion (Wong et al., 2018; Liu et al., 2014; Hu et al., 2012) . In this respect, the SARS-CoV

Papain-like proteasome (PL pro ), which, along with the main protease M pro (also called 3CL pro ), is essential for the cleavage and processing of the viral poly-protein, was found to repress IFNγ synthesis and secretion in lung cell cultures by targeting IRF3 phosphorylation and nuclear translocation (Devaraj et al., 2007; . Interestingly, PL pro was reported to have DUB activity in human cells by recognizing the LXGG consensus de-ubiquitination motif and by directly binding to the proteasome through the N-terminal Ub-like domain which is present in its structure (Ma et al., 2010) . Although the DUB activity was shown to be ineffective in altering the repertoire of host poly-Ub proteins, since Ub-conjugation is a post-translational mechanism of regulation of intracellular trafficking and receptor signalling, it would be relevant to uncover the link between IRF3 signalling and Ub-labelling by PL pro .

In this framework, viral accessory protein 3a was reported to promote IFN-I receptor ubiquitination and proteasome degradation (Minakshi et al., 2009) . Along with this, accessory proteins 8a and 8b or 8ab, which were found to be expressed either as a single protein or spliced protein in different viruses at different stages of SARS-CoV infection spread in 2003, were found to bind to intracellular ubiquitinated proteins through an extensively glycosylated functional domain which account for ubiquitin binding and ubiquitin conjugation . Interestingly, 8b and 8ab appears to stimulate the UPS-mediated degradation of IRF3 at later stages of virus replication than PL pro was supposed to do (Wong et al., 2018) . Remarkably, proteasome inhibition was found to be reveal that a major role for the UPS is to drive antigenic processing and inflammation activation especially in monocytes/macrophages (Silswal et al., 2017; Horan et al., 2013; Forget et al., 2005) .

Furthermore, proteasome dysfunction in alveolar type II epithelial cells is associated with ARDS in the alveolar space of Rpt3-KO mice (Sitaraman et al., 2019) . From the second point of view, besides the known contribution in modulating the transcriptional activity of nuclear factors, such as NF-kB (extensively discussed in paragraph 3.2.1), additional tissue-specific mechanisms which should intervene in coronavirus infection are expected to take part to the pathogenesis. In this regard, the clearance of specific proteins, such as elafin by the exceedingly activated UPS might be a relevant factor in sustaining inflammation. In fact, elafin is a serine-proteasome inhibitor and its inhibitory activity resides within the C-terminal domain which has specificity for NE and proteinase 3. Notably, transglutaminase substrate binding motif (GQDPVK) is present at the N-terminus which allows it to cross-link extracellular matrix proteins (Kerrin et al., 2013) .

In addition, clinical studies highlight that the 20S proteasome is released in the alveolar space during ARDS in an active configuration, and that immune-proteasome subunits are increased in the alveolar space envisaging a prognostic relevance of its quantification (Sixt et al., 2009; Sixt et al., 2012) . As a whole, the disastrous SARS-Cov-2 experience suggests that multi-faceted efforts by the scientific community are demanded to clarify the pathogenesis of coronaviruses especially in view of the concrete possibility that additional spill-overs in the next future might come up posing new pandemic threats. The central role of the UPS in regulating the complex dynamic of interactions between the pathogen and the host, along with the growing interest in the development of UPS modulators, could provide further clues for the identification of valid approaches which allow to limit the sanitarian, social and economic costs of similar pandemia.

The authors declare that there are no conflicts of interest.

Proteasome is a highly dynamic complex as demonstrated by the existence of alternative forms of proteasome which deal with specific biological roles. The immune-proteasome is the most studied alternative form of proteasome and its proteolytic activity has been long linked only to generation of antigenic peptides for MHC class I presentation Rousseau and Bertolotti, 2018) . However, a number of studies have reported a role for immune-proteasome in B and T cell differentiation, monocytes and dendritic cells activation, in the maintenance of pluripotency of stem cells (Atkinson et al., 2012) , in the differentiation of non-immune cells, such as skeletal muscle ones and also in the homeostasis of nervous cells (Kaur and Batra, 2016; Kimura et al., 2015) .

Along with this, immune-proteasome dysregulation has also been associated with various human diseases, including cancer, immune and inflammatory disorders: in fact, either hyper-activation or hypo-activation may turn out into a hyper-immune or hypo-immune phenotype (Eskandari et al., 2017) . Therefore, there has been a great effort to develop specific immune-proteasome inhibitors, which showed minimal cross-reactivity with constitutive proteasome. From the structural point of view, the immune-proteasome differs from the canonical 26S for the replacement of catalytic subunits with its immune highly homolog counterparts b1i (LMP2), b2i (MECL1), and b5i (LMP7) Sherman and Li, 2020) . Remarkably, these immune-proteasome subunits are constitutively expressed in different tissues, such as thymus and spleen. Moreover, non-immune cells preferentially incorporate them during the assembly de novo of 20S particles following exposition to proinflammatory stimuli (e.g., IFN-γ, TNF-α, and lipopolysaccharide) or cytokineindependent stressors (e.g., oxidative stress) (Griffin et al., 1998; Heink et al., 2005; Ferrington et al., 2012; Murata et al., 2018) . Immune-subunits incorporation has been proposed to proceed cooperatively, since the direct binding of β5i to chaperone POMP is followed by the quick recruitment of b1i and b2i. Therefore, the rate of their assembly is about four times faster than that of canonical subunits in the forming 20S, a finding consistent with the primary biological role of immune-proteasome, which is demanded to cope with patho-physiological challenges in a dynamic and highly efficient manner (Groettrup et al., 1997; Griffin et al., 1998; Murata et al., 2018) . The subunit substitution accounts for a shift in the catalytic preferences and activity; in fact, immuneproteasome exhibits elevated level of CT-L and T-L activities which favour the production of peptides with terminal basic or hydrophobic residues that fit better into the cleft of the MHC class I molecule (Rousseau and Bertolotti, 2018; Murata et al., 2018) .

IFN-γ also induces the expression of another important regulator, besides 19S, of 20S activity, the 11S regulator (PA28) (Sherman and Li, 2020; Cascio et al., 2001; .

Mammalian cells express three different subunits of 11S regulator: PA28α, PA28β, and PA28γ.

PA28α and PA28α assemble into a hetero-heptameric complex, primarily located in the cytoplasm, while homo-heptameric PA28γ is mainly present inside the nucleus (Wójcik et al., 1998; . Although the role of these regulators is not clear, it has been reported that both forms increase after oxidative stress, suggesting their involvement in the degradation of damaged proteins (Pickering et al., 2010; Pickering et al., 2012; Kors et al., 2019; Thibaudeau and Smith, 2018) .

Accordingly, PA28α-β association with the 20S does not enhance the degradation of polyubiquitinated protein/peptide substrates in vitro Lobanova et al., 2018) . In addition to its role in preserving cellular homeostasis after oxidative stress, the PA28 role in the regulation of the immune response has been extensively studied (Tanahashi et al., 1997; Früh and Yang, 1999; Preckel et al., 1999; . In fact, following INFγ stimulation, the level of PA28α-β binding to inducible 20S increases, enhancing its proteolytic activity and mediating the generation of antigenic peptides (Groettrup et al., 1996; Sijts et al., 2002; Fort et al., 2015) . Unlike PA28α-β, PA28γ is not induced by IFNγ, suggesting a different biological role for PA28γ-20S complex. Although the role of this complex remains elusive, a number of studies imply an involvement in the cell cycle progression (Kors et al., 2019) .

In addition to immune-proteasome, other tissue-specialized forms of proteasome are thymoproteasome and testis-proteasome (spermato-proteasome). The first one is expressed by cortical thymic epithelial cells and contains two immune catalytic subunits, B1i and B2i, and a thymus specific subunit (B5t), that, unlike β5 and β5i, is characterized by a number of hydrophilic amino acids in its catalytic pocket. Thus, thymo-proteasome produces a distinct spectrum of peptide fragments, and cells expressing it display a unique set of peptides associated with MHC-I molecules (Murata et al., 2007; Florea et al., 2010; Sasaki et al., 2015) . Thymo-proteasome role accounts for the positive selection of developing T cells, since it is essential to optimize the release of the repertoire of peptides for CD8 + T cell (Nitta et al., 2010; Xing et al., 2013; Takada et al., 2015; Murata et al., 2018) .

Spermato-proteasome is a testis-specific form of proteasome (described exclusively in spermatocytes, spermatids and sperm), and it is characterized by a chronologically defined expression. It contains a specific α4 subunit (α4s) (PMSA8 gene) in the place of corresponding constitutive α-subunit, whose incorporation into a newly formed 20S is mutually exclusive and does not alter the catalytic preferences of the constitutive 20S Uechi et al., 2014;  J o u r n a l P r e -p r o o f Morozov and Karpov, 2019) . Elevated expression of PSMA8 has been identified in different tumours, such as large B-cell lymphoma, thymoma, and testicular germ cell tumours. However, the biological significance and the possibility to make α4s a therapeutic target candidate has not been elucidated yet (Bruggerman et al., 2018; Morozov and Karpov, 2019) . The incorporation of α4s in spermato-proteasome seems to favour the 20S association with another gate-activating RP, namely PA200 (Blm10 in yeast) . PA200 is a nuclear-specific proteasome activator and it is expressed in all mammalian tissues, but it is particularly abundant in the testis, where it plays a crucial role in spermatogenesis (Ustrell et al., 2005; Khor et al., 2006) . Accordingly, PA200

deletion markedly reduces fertility of male mice (Khor et al., 2006) , and the PA200/20S spermatoproteasome complex catalyses ubiquitin-independent degradation of acetylated core histones during DNA repair and spermatogenesis . PA200 also binds constitutive proteasome and the amount of PA200/constitutive 20S, as well as of PA28/20S complex, increases upon 26S

inhibition, contributing to adapt the pool of different proteasome populations to the cell condition (Welk et al., 2016) . The crystal structure of this complex has revealed that C-terminal HbYX motif in PA200 fits between α5 and α6 inter-subunit pocket, mediating 20S gate opening (Sadre-Bazzaz et al., 2010; Witkowska et al., 2017) . Interestingly, it strongly stimulates the rate of C-L activity, although its biological significance is poorly clear yet (Ustrell et al., 2002) . Recently, it has been reported the identification of a non-canonical variant of constitutive 20S in mammalian cells, previously identified in yeast. It is known as alternative 'α4-α4' proteasome, which assembles upon replacement of α3 with an additional α4 subunit in the position normally occupied by the former (Velichutina et al., 2004; Kusmierczyk et al., 2008; Padmanabhan et al., 2016) . Importantly, mammalian cells, primed to assemble these alternative proteasomes, exhibit enhanced resistance to cellular stress induced by metal ions (Padmanabhan et al., 2016) .

The existence of interchangeable subunits, and thus, of alternative proteasome forms above described, as well as the identification of hybrid proteasome particles (i.e., ) whose biological function is poorly known, underlie how cells modify proteasome repertoire in relation to its specific need (Cascio et al., 2002; Morozov and Karpov, 2019; Thibaudeau and Smith, 2019) .

De-ubiquitinases (DUBs), which catalyse ubiquitin moieties removal from target proteins, are key components of the UPS, being involved in ubiquitin recycling and editing (Yuan et al. 2018) . Three

DUBs are associated with the proteasome: Rpn11, a Zn 2+ metallo-protease, which is part of the lid, J o u r n a l P r e -p r o o f USP14 and Uch37 which are two cysteine proteases, extrinsically associated with the base (see section 2.3.1) (D'Arcy et al., 2015) . Several studies have proposed that de-ubiquitination by Rpn11 stimulates the substrate degradation by removing bulky ubiquitin chain that otherwise might impair further substrate translocation into the proteasome (Poot et al., 2017) ; on the other hand, the ATP/independent de-ubiquitination by Usp14 and Uch37 is envisaged to suppress substrate degradation, promoting its premature dissociation from the proteasome (Lam et al., 1997; Hanna et al., 2006; Lee et al., 2016b) . Among the three DUBs, Usp14 is the most attractive way of intervention to regulate proteasome activity (Wertz and Murray, 2019; Chakraborty et al., 2018) . Human USP14 consists of two domains, namely (i) a N-terminal ubiquitin-like domain (UBL) and (ii) a C-terminal DUB domain, which contains the catalytic triad, Cys114, His435, and Asp451. In the free unbound state, the catalytic domain of Usp14 is characterized by a low level of de-ubiquitinase activity, whereas when it binds proteasome its activity is increased about 800-fold and shows a preference for substrates ubiquitinated at more than one site (Koulich et al., 2008; Hu et al., 2005; Lee et al., 2016b) . It has been shown that Usp14 inhibition stimulates the degradation of some specific proteasome substrates in mammalian cells, such as cancer and neurodegeneration related proteins Homma et al., 2015; Zhu et al., 2016; McKinnon et al., 2016; Boselli et al., 2017; Liao et al., 2017; Poot et al., 2017) . Additionally, Usp14 activation through phosphorylation of Ser432 residue by AKT results in the suppression of the degradation of short-lived proteins, that may in turn promote tumour cell proliferation (Xu et al., 2015; Wei et al., 2017; Kim and Goldberg, 2017) . Some evidences also suggest that Usp14 expression is closely related to the onset of different tumours, including breast, gastric and lung cancer (Wu et al., 2013; Zhu et al., 2016 (b) ; Fu et al., 2018) and its activity is required for nervous system development and functioning (Wilson et al., 2002; Chen et al., 2011; Vaden et al., 2015; Kiprowska et al., 2017) . Thus, considerable efforts have been dedicated to the discovery of small molecules that functionally inhibit Usp14 and several ones have been identified, such as b-AP15, auranofin, WP1130 and curcumin analogue AC17 (Kapuria et al., 2010; Zhou et al., 2013; Wang et al., 2014; Coughlin et al., 2014; Liu et al., 2014; D'Arcy et al., 2015; Xia et al., 2019; Wertz and Murray, 2019) . A common feature of most of these compounds is the presence of α,β-unsaturated carbonyl groups which can form covalent adducts with free thiols in the active site by Michael addition (D'Arcy et al., 2015; . Unfortunately, these compounds usually have poor selectivity across the DUB family, since most of them are cysteine enzymes which are easily "druggable" by compounds containing Michael acceptors (D'Arcy et al., 2015) . The small molecule IU1 was the first specific inhibitor identified, exhibiting excellent selectivity for USP14 over other DUBs . Co-crystal studies reveal J o u r n a l P r e -p r o o f a unique mechanism of action of IU1 that exerts its inhibitory activity by binding to the thumb-palm cleft region of Usp14 catalytic domain, sterically preventing ubiquitin binding to the C-terminal of Usp14 . Interestingly, the prion protein shows accelerated degradation upon IU1 treatment, and a more powerful analogue (IU1-47) enhances tau degradation in neurons (Homma et al., 2015; McKinnon et al., 2016; Boselli et al., 2017) , rendering it an intriguing target also for neurodegenerative diseases. VLX1570 is an analogue of b-AP15, being characterized by a higher potency and an improved solubility, which shows consistent anti-tumour activity in orthotopic and xenograft models of MM, lymphoma, Ewing's sarcoma, and other malignancies (D'Arcy et al., 2014; Chitta et al., 2015; Shukla et al., 2016) . Moreover, VLX1570 retains prominent activity in bortezomib-resistant MM cells (Shukla et al., 2016; Rowinsky et al., 2020) .

These studies together with the good tolerability profile, reported in preclinical models, provided the rationale for investigating this drug in clinical trials on patients with RRMM (Rowinsky et al., 2020) . Thus, in a phase 1 study fourteen patients with RRMM were enrolled and treated with escalating doses of intravenous infusion of VLX1570 ranging from 0.05 to 1.2 mg/kg and antimyeloma effects were observed at dose of 0.6 mg/kg or more. Unfortunately, two patients treated with 1.2 mg/kg dose experienced severe and progressive respiratory insufficiency, culminating in death; thus, due to severity of the toxicity, the study was discontinued (Rowinsky et al., 2020) .

Beside this molecule, no other inhibitors targeting DUBs have entered into clinical trial so far.

However, since they are abnormally expressed in a variety of tumours and/or in tumour microenvironment (Yuan et al. 2018) , making them ideal anticancer target candidates, the identification of selective small-molecule inhibitors for Usp14 and in general for other specific DUBs remains an active and extremely challenging task.

The regulation of metabolic control of proteasome function is a challenging point in proteasome biology, which deserves particular attention, although many aspects are obscures yet. The coordinated balance of the two post-translational modifications, i.e., O-linked N-acetylglucosamine (O-GlcNAcylation) and phosphorylation appears to be crucial in this process (Zhang et al., 2003; Zhang et al., 2007; Rousseau and Bertolotti, 2018) . proteins, by using as substrate a product of hexosamine biosynthesis, the UDP-GlcNAc, whose availability is influenced by nutritional conditions. In fact, an increase in glucose availability raises UDP-GlcNAc levels and consequently promotes protein O-GlcNAcylation (Comer and Hart, 2000; Zachara and Hart, 2004) . It has been reported that 26S proteasome function is inhibited by the addiction of sugar moieties to the Rpt2 subunit (Zhang et al., 2003) , a finding which provides a link between glucose metabolism and protein turnover . In the liver and muscle tissues of mouse models, when blood glucose drops (e.g., under fasting conditions or during exercise) the PKA increase stimulates 26S activity selectively toward the clearance of short-lived regulatory proteins, with no alterations in proteasome content, confirming that proteasome activation occurs through post-synthetic modification of already existing particles (Rousseau and Bertolotti, 2018; VerPlank et al., 2019) . The low level of glucose also reduces the entry of this mono-saccharide into the hexosamine pathway, limiting the availability of UDP-GlcNAc; as a consequence, the O-GlcNAc modification of Rpt2 decreases thus removing the signal that inhibits proteasome function (Zhang et al., 2003; Zhang et al., 2007) . Moreover, it has been shown that lack of nutrient rapidly inhibits the stress and nutrient response of the mTOR complex. It results into the enhancement of autophagic process and of the rate of long-lived protein ubiquitination and, therefore, of their degradation by UPS . All these events bring to a general activation of proteolysis, promoting cellular adaptation, facilitating damaged and potentially toxic protein clearance, and providing essential amino acids for the synthesis of proteins necessary for cell survival and energy production VerPlank and Goldberg, 2015; Rosseau and Bertolotti, 2016) . A. Structure of the 20S proteasome particle as viewed from the top (top panel) or the side (bottom panel). The protein backbone of the subunits is presented as ribbon. B. Active site a threonine peptidase subunit (β5) of the proteasome. The protein backbone of the β5 subunit is represented as turquoise ribbon, catalytic residue (Thr1) and other residues that helps maintains the structural stability of the catalytic site (Lys33, Asp17, Ser129, Asp166 and Ser169) are represented as sticks. Polar interactions are indicated as black dashed lines together with the corresponding distances. C. left: Schematic representation of the relative orientation of the subunits of the 20S particle. right: vertical cross-section of the 19S particle, the α-subunit rings are represented as red ribbons, the βsubunit rings as blue ribbons, the outline of the internal cavity and the internal "chambers" are highlighted with a black dashed line. J o u r n a l P r e -p r o o f levels are also modulated through the ubiquitin-dependent degradation by the 26S, and further by a ubiquitin independent pathway by the 20S.

Figure legend is restricted to NF-kB and p53, whose mechanisms of transcription induction is not sketched. PIs stands for Proteasome Inhibitors and the red arrows indicate the steps of NF-kB, p53 and p21 turn-over which are blocked by this class of drugs. ; are reported. The β-5 subunit is represented as turquoise ribbon, the β-1 subunit is represented as purple ribbon, the inhibitors are represented as orange sticks and the protein residues interacting with the inhibitors as grey sticks. Alkalay, I., Yaron, A., Hatzubai, A., Orian, A., Ciechanover, A., & Ben-Neriah, Y. (1995) . Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway. Proceedings of the National Academy of Sciences of the United States of America, 92 (23) Bigelow, S., Hough, R., & Rechsteiner, M. (1981) . The selective degradation of injected proteins occurs principally in the cytosol rather than in lysosomes. Cell, 25 (1) Thomas, S. N., Jeng, Y., Yang, F., Davies, P., & Yang, A. J. (2006) . Alzheimer diseasespecific conformation of hyperphosphorylated paired helical filament-Tau is polyubiquitinated through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation. The Journal of Biological Chemistry, 281 (16) Dahlqvist, J., Klar, J., Tiwari, N., Schuster, J., Törmä, H., Badhai, J., Pujol, R., van Steensel, M. A. M., Brinkhuizen, T., Brinkhuijzen, T., Gijezen, L., Chaves, A., Tadini, G., Vahlquist, A., & Dahl, N. (2010) . A single-nucleotide deletion in the POMP 5' UTR causes a transcriptional switch and altered epidermal proteasome distribution in KLICK genodermatosis. American Journal of Human Genetics, 86 (4) J o u r n a l P r e -p r o o f de Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R., & Appelmans, F. (1955) . Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochemical Journal, 60 (4) Drach, J., Huang, H., Samoilova, O., Belch, A., Farber, C., Bosly, A., Novak, J., Zaucha, J., Dascalescu, A., Bunworasate, U., Masliak, Z., Vilchevskaya, K., Robak, T., Pei, L., Rooney, B., van de Velde, H., & Cavalli, F. (2018) . Efficacy and safety of frontline rituximab, cyclophosphamide, doxorubicin and prednisone plus bortezomib (VR-CAP) or vincristine (R-CHOP) in a subset of newly diagnosed mantle cell lymphoma patients medically eligible for transplantation in the randomized, phase 3 LYM-3002 study. Leukemia & Lymphoma, 59 (4) Eytan, E., Ganoth, D., Armon, T., & Hershko, A. (1989) . ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin. Proceedings of the National Academy of Sciences of the United States of America, 86 (20) Leonardi, G., Rizzo, M., Falcone, A. P., Gottardi, D., … Boccadoro, M. (2010) . Bortezomibmelphalan-prednisone-thalidomide followed by maintenance with bortezomib-thalidomide compared with bortezomib-melphalan-prednisone for initial treatment of multiple myeloma: a randomized controlled trial. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, 28 (34) Takahashi, M., Kitaura, H., Kakita, A., Kakihana, T., Katsuragi, Y., Nameta, M., Zhang, L., Iwakura, Y., Nawa, H., Higuchi, M., Komatsu, M., & Fujii, M. (2018) . USP10 Is a Driver of Ubiquitinated Protein Aggregation and Aggresome Formation to Inhibit Apoptosis. IScience, 9, 433-450. https://doi.org/10.1016 IScience, 9, 433-450. https://doi.org/10. /j.isci.2018 Tanahashi, N., Yokota, K., Ahn, J. Y., Chung, C. H., Fujiwara, T., Takahashi, E., DeMartino, G. N., Slaughter, C. A., Toyonaga, T., Yamamura, K., Shimbara, N., & Tanaka, K. (1997) . Molecular properties of the proteasome activator PA28 family proteins and γ-interferon regulation. Genes to Cells, 2(3), 195-211. https://doi.org/10.1046/j. 1365 -2443 .d01-308.x Tanaka, K, Waxman, L., & Goldberg, A. L. (1983 . ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin. The Journal of Cell Biology, 96 (6) Tang, X., Wu, C., Li, X., Song, Y., Yao, X., Wu, X., Duan, Y., Zhang, H., Wang, Y., Qian, Z., Cui, J., & Lu, J. (n.d.) . On the origin and continuing evolution of SARS-CoV-2. National Science Review. https://doi.org/10.1093/nsr/nwaa036 Tanji, K., Miki, Y., Maruyama, A., Mori, F., Mimura, J., Itoh, K., Kamitani, T., & Wakabayashi, K. (2016) . The role of NUB1 in α-synuclein degradation in Lewy body disease model mice. Tanji, K., Mori, F., Mimura, J., Itoh, K., Kakita, A., Takahashi, H., & Wakabayashi, K. (2010) . Proteinase K-resistant alpha-synuclein is deposited in presynapses in human Lewy body disease and A53T alpha-synuclein transgenic mice. Acta Neuropathologica, 120(2), 145-154. https://doi.org/10.1007/s00401-010-0676-z Tarohda, T., Yamamoto, M., & Amamo, R. (2004). Regional distribution of manganese, iron, copper, and zinc in the rat brain during development. Analytical and Bioanalytical Chemistry, 380 (2) 

Development of peptide-based reversing agents for p-glycoprotein-mediated resistance to carfilzomib

Efficacy of continuous, daily, oral, ultra-low-dose 200 mg acyclovir to prevent herpes zoster events among bortezomib-treated patients: a report from retrospective study

Nonproteasomal targets of the proteasome inhibitors bortezomib and carfilzomib: a link to clinical adverse events

Molecular modeling on porphyrin derivatives as β5 subunit inhibitor of 20S proteasome

Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly

Assembly of the 26 S complex that degrades proteins ligated to ubiquitin is accompanied by the formation of ATPase activity

Interrelation between protein synthesis, proteostasis and life span

Updated survival analysis of a randomized phase III study of subcutaneous versus intravenous bortezomib in patients with relapsed multiple myeloma

Identity of the 19S "prosome" particle with the large multifunctional protease complex of mammalian cells (the proteasome)

PKA rapidly enhances proteasome assembly and activity in in vivo canine hearts

Structure and Function of the 26S Proteasome

Superiority of tandem autologous transplantation over standard therapy for previously untreated multiple myeloma

Dual functions of the Hsm3 protein in chaperoning and scaffolding regulatory particle subunits during the proteasome assembly

Spectrum and functional validation of PSMB5 mutations in multiple myeloma

The immunoproteasome: a novel drug target for autoimmune diseases

PNPLA3-I148M: a problem of plenty in non-alcoholic fatty liver disease

Proteasomes can degrade a significant proportion of cellular proteins independent of ubiquitination

Electron microscopy and image analysis of the multicatalytic proteinase

Phosphodiesterase 10A Inhibition Improves Cortico-Basal Ganglia Function in Huntington's Disease Models

Near-atomic resolution structural model of the yeast 26S proteasome

Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase

Promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing

Natural polyphenols as proteasome modulators and their role as anti-cancer compounds

Safety of ixazomib for the treatment of multiple myeloma

20S proteasome and its inhibitors: crystallographic knowledge for drug development

A protein catalytic framework with an N-terminal nucleophile is capable of self-activation

Cell death in the nervous system

Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production

Spreading of pathology in neurodegenerative diseases: a focus on human studies

Massive expression of germ cell-specific genes is a hallmark of cancer and a potential target for novel treatment development

Bortezomib-induced acute interstitial nephritis. Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association -European Renal Association

Protein Misfolding, Functional Amyloid, and Human Disease

mTORC1 accelerates retinal development via the immunoproteasome

Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation

Proteasome inhibition induces a senescence-like phenotype in primary human fibroblasts cultures

Overexpression of hUMP1/POMP proteasome accessory protein enhances proteasome-mediated antioxidant defence

Proteasome Function Determines Cellular Homeostasis and the Rate of Aging

Anti-ageing and rejuvenating effects of quercetin

The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg

Mechanisms of ubiquitin-mediated, limited processing of the NF-kappaB1 precursor protein p105

ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation

Protein Quality Control by Molecular Chaperones in Neurodegeneration

Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition. ELife, 5, e13027

Organic copper complexes as a new class of proteasome inhibitors and apoptosis inducers in human cancer cells

The ubiquitin-proteasome system in neurodegenerative diseases: precipitating factor, yet part of the solution

Synergistic anti-myeloma activity of the proteasome inhibitor marizomib and the IMiD® immunomodulatory drug pomalidomide

Essential fatty acids in Huntington's disease

Reduced Levels of Proteasome Products in a Mouse Striatal Cell Model of Huntington's Disease

Electrostatic Map Of Proteasome α-Rings Encodes The Design of Allosteric Porphyrin-Based Inhibitors Able To Affect 20S Conformation By Cooperative Binding

Proteasomal degradation of tau protein

Protein modification by oxidants and the role of proteolytic enzymes

Degradation of oxidized proteins by the 20S proteasome

A Set of Activity-Based Probes to Visualize Human (Immuno)proteasome Activities

Functions of lysosomes

Enzymic content of the mitochondria fraction

A 26 S protease subunit that binds ubiquitin conjugates

Marizomib activity as a single agent in malignant gliomas: ability to cross the blood-brain barrier

Inhibition of 26S proteasome activity by huntingtin filaments but not inclusion bodies isolated from mouse and human brain

Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants

The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins

Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain

Oral ixazomib maintenance following autologous stem cell transplantation (TOURMALINE-MM3): a double-blind, randomised

Multicenter phase II study of bortezomib in patients with relapsed or refractory mantle cell lymphoma

Activity-based profiling reveals reactivity of the murine thymoproteasome-specific subunit beta5t

Multicatalytic Proteinase in Fish Muscle

A balance of protein synthesis and proteasome-dependent degradation determines the maintenance of LTP

Proteasome-mediated degradation of STAT1alpha following infection of macrophages with Leishmania donovani

The pore of activated 20S proteasomes has an ordered 7-fold symmetric conformation

Structural determinants for the intracellular degradation of human thymidylate synthase

Evolution of proteasome regulators in eukaryotes

Peptide therapeutics: current status and future directions

The proteasome maturation protein POMP facilitates major steps of 20S proteasome formation at the endoplasmic reticulum

The combination of bendamustine, bortezomib, and rituximab for patients with relapsed/refractory indolent and mantle cell non-Hodgkin lymphoma

Antigen presentation by MHC class I and its regulation by interferon gamma

A first in human phase I study of the proteasome inhibitor CEP-18770 in patients with advanced solid tumours and multiple myeloma

Parkin functions as an E2-dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicleassociated protein, CDCrel-1

Cytosolic PINK1 promotes the targeting of ubiquitinated proteins to the aggresome-autophagy pathway during proteasomal stress

Superiority of the triple combination of bortezomib-thalidomide-dexamethasone over the dual combination of thalidomidedexamethasone in patients with multiple myeloma progressing or relapsing after autologous transplantation: the MMVAR/IFM 2005-04 Randomized Phase III Trial from the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation

Drug combinations with proteasome inhibitors in antitumor therapy

A Phase Ib/II Study of Oprozomib in Patients with Advanced Multiple Myeloma and Waldenström Macroglobulinemia

Inhibition of the striatal specific phosphodiesterase PDE10A ameliorates striatal and cortical pathology in R6/2 mouse model of Huntington's disease

Phase II 2-arm trial of the proteasome inhibitor, PS-341 (bortezomib) in combination with irinotecan or PS-341 alone followed by the addition of irinotecan at time of progression in patients with locally recurrent or metastatic squamous cell carcinoma of the head and neck (E1304): a trial of the Eastern Cooperative Oncology Group

Angiogenesis and Multiple Myeloma

Exposure of hydrophobic moieties promotes the selective degradation of hydrogen peroxide-modified hemoglobin by the multicatalytic proteinase complex, proteasome

Proline-and Arginine-Rich Peptides as Flexible Allosteric Modulators of Human Proteasome Activity

Proline-and Arginine-Rich Peptides as Flexible Allosteric Modulators of Human Proteasome Activity

Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein

The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction

The propagation of prion-like protein inclusions in neurodegenerative diseases

Degradation of abnormal proteins in Escherichia coli (protein breakdownprotein structure-mistranslation-amino acid analogs-puromycin)

Intracellular protein degradation in mammalian and bacterial cells

Intracellular protein degradation in mammalian and bacterial cells: Part 2

Blocking Cancer Growth with Less POMP or Proteasomes

The ubiquitinproteasome system: Potential therapeutic targets for alzheimer's disease and spinal cord injury

Associations of human retinal very long-chain polyunsaturated fatty acids with dietary lipid biomarkers

Bortezomib in patients with relapsed or refractory mantle cell lymphoma: updated time-to-event analyses of the multicenter phase 2 PINNACLE study

Phase II study of proteasome inhibitor bortezomib in relapsed or refractory B-cell non-Hodgkin's lymphoma

Second Generation Proteasome Inhibitors in Multiple Myeloma. Anti-Cancer Agents in Medicinal Chemistry

The cationic porphyrin TMPyP4 down-regulates c-MYC and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo

Patterns of cardiac toxicity associated with irreversible proteasome inhibition in the treatment of multiple myeloma

Immunoproteasome assembly: cooperative incorporation of interferon gamma (IFNgamma)-inducible subunits

The 26S proteasome is a multifaceted target for anti-cancer therapies

The subunits MECL-1 and LMP2 are mutually required for incorporation into the 20S proteasome

A gated channel into the proteasome core particle

A gated channel into the proteasome core particle

Crystal structure of the boronic acidbased proteasome inhibitor bortezomib in complex with the yeast 20S proteasome

Structure of 20S proteasome from yeast at 2.4 A resolution

The catalytic sites of 20S proteasomes and their role in subunit maturation: a mutational and crystallographic study

Substrate access and processing by the 20S proteasome core particle

Probing structural determinants distal to the site of hydrolysis that control substrate specificity of the 20S proteasome

Degradation of oxidized proteins in K562 human hematopoietic cells by proteasome

HSP70 mediates dissociation and reassociation of the 26S proteasome during adaptation to oxidative stress

Amyloid fibril formation by an SH3 domain

Glutamate stimulates glutamate receptor interacting protein 1 degradation by ubiquitin-proteasome system to regulate surface expression of GluR2

Retinal Anatomy and Pathology

Effects of Strong CYP3A Inhibition and Induction on the Pharmacokinetics of Ixazomib, an Oral Proteasome Inhibitor: Results of Drug-Drug Interaction Studies in Patients With Advanced Solid Tumors or Lymphoma and a Physiologically Based Pharmacokinetic Analysis

Clinical Pharmacology of Ixazomib: The First Oral Proteasome Inhibitor

A phase I study to assess the mass balance, excretion, and pharmacokinetics of [14C]-ixazomib, an oral proteasome inhibitor, in patients with advanced solid tumors

Switching from body surface area-based to fixed dosing for the investigational proteasome inhibitor ixazomib: a population pharmacokinetic analysis

Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome

Hemin inhibits ATP-dependent ubiquitin-dependent proteolysis: Role of hemin in regulating ubiquitin conjugate degradation

Redundant Roles of Rpn10 and Rpn13 in Recognition of Ubiquitinated Proteins and Cellular Homeostasis

A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes

Preliminary report: inhibition of cellular proteasome activity by free fatty acids

Activity-dependent growth of new dendritic spines is regulated by the proteasome

Oral delivery of peptide drugs: barriers and developments

Direct cellular delivery of human proteasomes to delay tau aggregation

Epoxomicin, a new antitumor agent of microbial origin

The hallmarks of cancer

A ubiquitin stress response induces altered proteasome composition

An inducible chaperone adapts proteasome assembly to stress

TAK-063, a Novel Phosphodiesterase 10A Inhibitor, Protects from Striatal Neurodegeneration and Ameliorates Behavioral Deficits in the R6/2 Mouse Model of Huntington's Disease

Proteostasis in Huntington's disease: disease mechanisms and therapeutic opportunities

Proteasome inhibitors mechanism; source for design of newer therapeutic agents

Healthcare resource utilization with ixazomib or placebo plus lenalidomide-dexamethasone in the randomized, double-blind, phase 3 TOURMALINE-MM1 study in relapsed/refractory multiple myeloma

Oprozomib in patients with newly diagnosed multiple myeloma

Efficacy and safety results from a phase 1b/2, multicenter, open-label study of oprozomib and dexamethasone in patients with relapsed and/or refractory multiple myeloma

Bortezomib plus dexamethasone is superior to vincristine plus doxorubicin plus dexamethasone as induction treatment prior to autologous stemcell transplantation in newly diagnosed multiple myeloma: results of the IFM 2005-01 phase III trial

Release of a macromolecular protein component from human erythrocyte ghosts

Phase I Clinical Trial of Marizomib (NPI-0052) in Patients with Advanced Malignancies Including Multiple Myeloma: Study NPI-0052-102 Final Results

Crystal structure of the human 20S proteasome in complex with carfilzomib

Cycling of O-linked β-N -acetylglucosamine on nucleocytoplasmic proteins

Converging concepts of protein folding in vitro and in vivo

Molecular chaperones in protein folding and proteostasis

The structure of the 26S proteasome subunit Rpn2 reveals its PC repeat domain as a closed toroid of two concentric α-helical rings

Phosphodiesterase inhibition and modulation of corticostriatal and hippocampal circuits: Clinical overview and translational considerations

Perturbations of Ubiquitin-Proteasome-Mediated Proteolysis in Aging and Alzheimer's Disease. Frontiers in Aging Neuroscience, 11

The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing

IFN-gamma-induced immune adaptation of the proteasome system is an accelerated and transient response

Effect of cytochrome P450 3A4 inducers on the pharmacokinetic, pharmacodynamic and safety profiles of bortezomib in patients with multiple myeloma or non-Hodgkin's lymphoma

Phosphorylation of Tyr-950 in the proteasome scaffolding protein RPN2 modulates its interaction with the ubiquitin receptor RPN13

A novel role for the immunoproteasome in retinal function

Ubiquitin-specific protease-14 reduces cellular aggregates and protects against mutant huntingtin-induced cell degeneration: involvement of the proteasome and ER stress-activated kinase IRE1α

The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome

A first-in-human dose-escalation study of the oral proteasome inhibitor oprozomib in patients with advanced solid tumors

Loss of the Parkinson's disease-linked gene DJ-1 perturbs mitochondrial dynamics

Monoubiquitination of RPN10 regulates substrate recruitment to the proteasome

Relationship between cognitive impairment and retinal morphological and visual functional abnormalities in Alzheimer disease

Therapeutic antibodies for multiple myeloma

Effects of linoleic acid and cations on the activity of a novel high-molecular weight protease, ingensin, from human placent

A phase 2 study of two doses of bortezomib in relapsed or refractory myeloma

CRISPR-Cas9-based treatment of myocilin-associated glaucoma

Ocular MECP2 protein expression in patients with and without Rett syndrome

Treatment outcome with the combination of carfilzomib, lenalidomide, and low-dose dexamethasone (CRd) for newly diagnosed multiple myeloma (NDMM) after extended follow-up

Randomized phase 2 study: elotuzumab plus bortezomib/dexamethasone vs bortezomib/dexamethasone for relapsed/refractory MM

Defective autophagy in Parkinson's disease: role of oxidative stress

CaMKII, but not protein kinase A, regulates Rpt6 phosphorylation and proteasome activity during the formation of longterm memories

Interaction between pathogenic proteins in neurodegenerative disorders

Substrate receptors of proteasomes

Docosahexaenoic acid induces the degradation of HPV E6/E7 oncoproteins by activating the ubiquitin-proteasome system

Mutated myocilin and heterozygous Sod2 deficiency act synergistically in a mouse model of open-angle glaucoma

The ubiquitin-proteasome system: opportunities for therapeutic intervention in solid tumors

Proteasome Activation to Combat Proteotoxicity

Proteasomal Degradation of RPN4 via Two Distinct Mechanisms, Ubiquitin-dependent and -independent

Dynamic recruitment of ubiquitin to mutant huntingtin inclusion bodies

Expanded polyglutamine-containing N-terminal huntingtin fragments are entirely degraded by mammalian proteasomes

Detection of ubiquitinated huntingtin species in intracellular aggregates

Discovery, Development, and clinical applications of bortezomib

Parkin promotes proteasomal degradation of synaptotagmin IV by accelerating polyubiquitination

Chemical proteasome inhibition as a novel animal model of inner retinal degeneration in rats

The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor

Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein

Primary treatment of light-chain amyloidosis with bortezomib, lenalidomide, and dexamethasone

The olive constituent oleuropein exhibits proteasome stimulatory properties in vitro and confers life span extension of human embryonic fibroblasts

Betulinic acid, a natural PDE inhibitor restores hippocampal cAMP/cGMP and BDNF, improve cerebral blood flow and recover memory deficits in permanent BCCAO induced vascular dementia in rats

Emerging role of immunoproteasomes in pathophysiology

Proteasome inhibition by paired helical filament-tau in brains of patients with Alzheimer's disease

A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome

Impaired proteasome function in Alzheimer's disease

SARS coronavirus 8b reduces viral replication by down-regulating E via an ubiquitinindependent proteasome pathway

Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism

Proteolytic cleavage of elafin by 20S proteasome may contribute to inflammation in acute lung injury

Proteasome activator PA200 is required for normal spermatogenesis

Protein homeostasis in models of aging and age-related conformational disease

Targeted Disruption of the Myocilin Gene (Myoc) Suggests that Human Glaucoma-Causing Mutations Are Gain of Function

synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells

Modification by single ubiquitin moieties rather than polyubiquitination is sufficient for proteasomal processing of the p105 NF-kappaB precursor

Reduced basal autophagy and impaired mitochondrial dynamics due to loss of Parkinson's disease-associated protein DJ-1

20S proteasome biogenesis

Proteasome inhibitors -molecular basis and current perspectives in multiple myeloma

Proteasome: a Nanomachinery of Creative Destruction

Chargemediated proteasome targeting

Loss-less Nano-fractionator for High Sensitivity

Natural history of relapsed myeloma, refractory to immunomodulatory drugs and proteasome inhibitors: a multicenter IMWG study

Phase 2 trial of ixazomib in patients with relapsed multiple myeloma not refractory to bortezomib

Safety and tolerability of ixazomib, an oral proteasome inhibitor, in combination with lenalidomide and dexamethasone in patients with previously untreated multiple myeloma: an open-label phase 1/2 study

Ixazomib, lenalidomide, and dexamethasone in patients with newly diagnosed multiple myeloma: long-term follow-up including ixazomib maintenance

Randomized phase 2 trial of ixazomib and dexamethasone in relapsed multiple myeloma not refractory to bortezomib

Multiple myeloma

Ubiquitin proteasome system impairment, and L-Dopa response in zinc-induced Parkinsonism: resemblance to sporadic Parkinson's disease

An archaeal homolog of proteasome assembly factor functions as a proteasome activator

Assembly of the 20S proteasome

Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer

Crystal structure of human proteasome assembly chaperone PAC4 involved in proteasome formation

STEP61 is a substrate of the E3 ligase parkin and is upregulated in Parkinson's disease

Bortezomib combined with rituximab and dexamethasone is an active regimen for patients with relapsed and chemotherapy-refractory mantle cell lymphoma

The neurotoxicity of iron, copper and cobalt in Parkinson's disease through ROS-mediated mechanisms

Complete subunit architecture of the proteasome regulatory particle

Therapeutic peptides: Historical perspectives, current development trends, and future directions

Therapeutic peptides: Historical perspectives, current development trends, and future directions

20S proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals

Expression, post-translational modification and biochemical characterization of proteins encoded by subgenomic mRNA8 of the severe acute respiratory syndrome coronavirus

Proteasome inhibitor PS-341 inhibits human myeloma cell growth in vivo and prolongs survival in a murine model

Protein degradation by the ubiquitinproteasome pathway in normal and disease states

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies

Tau degradation: the ubiquitin-proteasome system versus the autophagy-lysosome system

H727 cells are inherently resistant to the proteasome inhibitor carfilzomib, yet require proteasome activity for cell survival and growth

Loss of Rpt5 protein interactions with the core particle and Nas2 protein causes the formation of faulty proteasomes that are inhibited by Ecm29 protein

A68: a major subunit of paired helical filaments and derivatized forms of normal Tau

Structural requirements within protoporphyrin IX in the inhibition of heat shock protein 90

iRhom1 regulates proteasome activity via PAC1/2 under ER stress

Tools to investigate the ubiquitin proteasome system

Multiple associated proteins regulate proteasome structure and function

Ecm29 fulfils quality control functions in proteasome assembly

Role of Proteasome Inhibitors in Relapsed and/or Refractory Multiple Myeloma

Patient-reported health-related quality of life from the phase III TOURMALINE-MM1 study of ixazomib-lenalidomide-dexamethasone versus placebolenalidomide-dexamethasone in relapsed/refractory multiple myeloma

Review. Clinical pharmacokinetics of bortezomib

Marizomib irreversibly inhibits proteasome to overcome compensatory hyperactivation in multiple myeloma and solid tumour patients

Boronic acid-containing proteasome inhibitors: alert to potential pharmaceutical bioactivation

The Nuclear Factor (Erythroid-derived 2)-like 2 and Proteasome Maturation Protein Axis Mediate Bortezomib Resistance in Multiple Myeloma

Liberation of functional p53 by proteasome inhibition in human papilloma virus-positive head and neck squamous cell carcinoma cells promotes apoptosis and cell cycle arrest

Nucleotide-dependent switch in proteasome assembly mediated by the Nas6 chaperone

Binding States of Protein-Metal Complexes in Cells

Enhancement of proteasome function by PA28α overexpression protects against oxidative stress

Correlation between TGF-β1 expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain-like protease

Angiotensinconverting enzyme 2 is a functional receptor for the SARS coronavirus

A new gold(I) complex-Au(PPh3)PT is a deubiquitinase inhibitor and inhibits tumor growth

Inhibiting the ubiquitinproteasome system leads to preferential accumulation of toxic N-terminal mutant huntingtin fragments

The Parkinson's disease protein LRRK2 impairs proteasome substrate clearance without affecting proteasome catalytic activity

Proteotoxic stress induces phosphorylation of p62/SQSTM1 by ULK1 to regulate selective autophagic clearance of protein aggregates

A Sentinel in the Crosstalk Between the Nervous and Immune System: The (Immuno)-Proteasome

Regulation of feedback between protein kinase A and the proteasome system worsens Huntington's disease

Atypical ubiquitination by E3 ligase WWP1 inhibits the proteasome-mediated degradation of mutant huntingtin

Leucine-rich repeat kinase 2 regulates the progression of neuropathology induced by Parkinson's-disease-related mutant alpha-synuclein

CIP2A-mediated Akt activation plays a role in bortezomib-induced apoptosis in head and neck squamous cell carcinoma cells

Relationship between beta-amyloid degradation and the 26S proteasome in neural cells

The ubiquitin-proteasome cascade is required for mammalian longterm memory formation

Pharmacokinetics and safety of bortezomib in patients with advanced malignancies and varying degrees of liver dysfunction: phase I NCI Organ Dysfunction Working Group Study NCI-6432

Copper, iron and zinc in Alzheimer's disease senile plaques

Revealing the dynamics of the 20 S proteasome phosphoproteome: a combined CID and electron transfer dissociation approach

Health-related quality of life in the ENDEAVOR study: carfilzomib-dexamethasone vs bortezomib-dexamethasone in relapsed/refractory multiple myeloma

Herp Promotes Degradation of Mutant Huntingtin: Involvement of the Proteasome and Molecular Chaperones

20S proteasome and glyoxalase 1 activities decrease in erythrocytes derived from Alzheimer's disease patients

The proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents

Proteasome inhibition in vivo promotes survival in a lethal murine model of severe acute respiratory syndrome

c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

ClpX(P) generates mechanical force to unfold and translocate its protein substrates

Proteasome inhibitors in cancer therapy

Induction of cell death by the novel proteasome inhibitor marizomib in glioblastoma in vitro and in vivo

Catalytic mechanism and assembly of the proteasome

Dynamic Regulation of the 26S Proteasome: From Synthesis to Degradation

Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding

Copper entry into human cells: progress and unanswered questions

Copper complexes as anticancer agents. Anti-Cancer Agents in Medicinal Chemistry

Overall survival with daratumumab, bortezomib, melphalan, and prednisone in newly diagnosed multiple myeloma (ALCYONE): a randomised, open-label, phase 3 trial

Bortezomib plus melphalan and prednisone in elderly untreated patients with multiple myeloma: updated time-to-events results and prognostic factors for time to progression

Bortezomib plus melphalan and prednisone in elderly untreated patients with multiple myeloma: results of a multicenter phase 1/2 study

Impact of prior therapy on the efficacy and safety of oral ixazomib-lenalidomide-dexamethasone vs

Bortezomib plus melphalan and prednisone compared with melphalan and prednisone in previously untreated multiple myeloma: updated follow-up and impact of subsequent therapy in the phase III VISTA trial

Chaperone-assisted assembly of the proteasome core particle

Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction

Neuroprotection by rat Müller glia against high glucose-induced neurodegeneration through a mechanism involving ERK1/2 activation

Aging and Neuronal Vulnerability

Conformational switching of the 26S proteasome enables substrate degradation

Triterpene derivatives that block entry of human immunodeficiency virus type 1 into cells

Emerging Developments in Targeting Proteotoxicity in Neurodegenerative Diseases

The ubiquitin-proteasome system in neurodegeneration

Impairment of the ubiquitin-proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures

Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson's disease

Retinal antigen-specific regulatory T cells protect against spontaneous and induced autoimmunity and require local dendritic cells

Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity

Coordination between proteasome impairment and caspase activation leading to TAU pathology: neuroprotection by cAMP

Selected cytotoxic gold compounds cause significant inhibition of 20S proteasome catalytic activities

Differential Protein Expression Profiles in Glaucomatous Trabecular Meshwork: An Evaluation Study on a Small Primary Open Angle Glaucoma Population

Cyclophosphamide-bortezomib-dexamethasone (CyBorD) produces rapid and complete hematologic response in patients with AL amyloidosis

Sigma-1 receptor is involved in degradation of intranuclear inclusions in a cellular model of Huntington's disease

The measurement of protein synthesis for assessing proteostasis in studies of slowed aging

The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor

CCT2 Mutations Evoke Leber Congenital Amaurosis due to Chaperone Complex Instability

Mechanisms of muscle wasting. The role of the ubiquitinproteasome pathway

The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications

Bortezomib, thalidomide, and dexamethasone with or without daratumumab before and after autologous stem-cell transplantation for newly diagnosed multiple myeloma (CASSIOPEIA): a randomised, open-label, phase 3 study. The Lancet

Phase 1/2 study of carfilzomib plus melphalan and prednisone in patients aged over 65 years with newly diagnosed multiple myeloma

Convenience, satisfaction, health-related quality of life of once-weekly 70 mg/m2 vs. twice-weekly 27 mg/m2 carfilzomib (randomized

Oral Ixazomib, Lenalidomide, and Dexamethasone for Multiple Myeloma

Once weekly versus twice weekly carfilzomib dosing in patients with relapsed and refractory multiple myeloma

Subcutaneous versus intravenous administration of bortezomib in patients with relapsed multiple myeloma: a randomised, phase 3, non-inferiority study

Once-weekly (70 mg/m2) vs twice-weekly (56 mg/m2) dosing of carfilzomib in patients with relapsed or refractory multiple myeloma: A post hoc analysis of the ENDEAVOR

KLICK syndrome: recognizable phenotype and hot-spot POMP mutation

Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging

The effect of aging on the mineral status of female mice

Proteasomes and Several Aspects of Their Heterogeneity Relevant to Cancer

Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice

The Parkinson's-associated protein DJ-1 regulates the 20S proteasome

Betulinic acid, a natural compound with potent anticancer effects

Modulation of protein quality control systems by food phytochemicals

Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination

Regulation of CD8+ T cell development by thymus-specific proteasomes

The immunoproteasome and thymoproteasome: functions, evolution and human disease

Immunoproteasome assembly and antigen presentation in mice lacking both PA28α and PA28β

Spotlight on ixazomib: potential in the treatment of multiple myeloma. Drug Design

Targeting the 26S Proteasome To Protect Against Proteotoxic Diseases

Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMP-PKA signaling

Lack of proteasome active site allostery as revealed by subunit-specific inhibitors

Light exposure induces ubiquitin conjugation and degradation activities in the rat retina

Ultrastructure of the human retina in aging and various pathological states

Targeting the ubiquitin-proteasome pathway to overcome anti-cancer drug resistance

Targeting the ubiquitin-proteasome pathway to overcome anti-cancer drug resistance

Endoplasmic reticulum stress activates autophagy but not the proteasome in neuronal cells: implications for Alzheimer's disease

DJ-1-binding compound B enhances Nrf2 activity through the PI3-kinase-Akt pathway by DJ-1-dependent inactivation of PTEN

Thymoproteasome shapes immunocompetent repertoire of CD8+ T cells

Can you teach old drugs new tricks?

Proteasome Inhibitors: Structure and Function

Genetic etiology of Parkinson disease associated with mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 genes: a mutation update

Patients with chemotherapy-refractory mantle cell lymphoma experience high response rates and identical progression-free survivals compared with patients with relapsed disease following treatment with single agent bortezomib: results of a multicentre Phase 2 clinical trial

A phase 1 dose escalation study of the safety and pharmacokinetics of the novel proteasome inhibitor carfilzomib (PR-171) in patients with hematologic malignancies

Phase II clinical experience with the novel proteasome inhibitor bortezomib in patients with indolent non-Hodgkin's lymphoma and mantle cell lymphoma

Macular versus Retinal Nerve Fiber Layer Parameters for Diagnosing Manifest Glaucoma: A Systematic Review of Diagnostic Accuracy Studies

Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein

An evidencebased review of ixazomib citrate and its potential in the treatment of newly diagnosed multiple myeloma

Synthesis and biological evaluation of thielocin B1 analogues as protein-protein interaction inhibitors of PAC3 homodimer

p62/SQSTM1 cooperates with Parkin for perinuclear clustering of depolarized mitochondria

Proteasome inhibitors for multiple myeloma

Posttranslational modifications and proteinopathies: how guardians of the proteome are defeated

Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions

Metalloenzyme-like activity of Alzheimer's disease beta-amyloid. Cu-dependent catalytic conversion of dopamine, cholesterol, and biological reducing agents to neurotoxic H(2)O(2)

Fludarabine, Bortezomib, Myocet and rituximab chemotherapy in relapsed and refractory mantle cell lymphoma

A multicatalytic protease complex from pituitary that forms enkephalin and enkephalin containing peptides

Regulation of the Peptidylglutamyl-Peptide Hydrolyzing Activity of the Pituitary Multicatalytic Proteinase Complex

Final overall survival results of a randomized trial comparing bortezomib plus pegylated liposomal doxorubicin with bortezomib alone in patients with relapsed or refractory multiple myeloma

Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies

Aneuploidy: implications for protein homeostasis and disease

Ubiquitin-proteasome system involvement in Huntington's disease

New Peptide-Based Pharmacophore Activates 20S Proteasome

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells

Association of melphalan and high-dose dexamethasone is effective and well tolerated in patients with AL (primary) amyloidosis who are ineligible for stem cell transplantation

Low Erythrocyte Levels of Proteasome and Acyl-Peptide Hydrolase (APEH) Activities in Alzheimer's Disease: A Sign of Defective Proteostasis

Bortezomib-melphalan-prednisone-thalidomide followed by maintenance with bortezomibthalidomide compared with bortezomib-melphalan-prednisone for initial treatment of multiple myeloma: updated follow-up and improved survival

Daratumumab, Bortezomib, and Dexamethasone for Multiple Myeloma

The productivity crisis in pharmaceutical R&D

Not4 E3 ligase contributes to proteasome assembly and functional integrity in part through Ecm29

Cotranslational assembly of proteasome subunits in NOT1-containing assemblysomes

Proteasome inhibitors induce p53-independent apoptosis in human cancer cells

Sodium diethyldithiocarbamate, an AIDS progression inhibitor and a copper-binding compound, has proteasome-inhibitory and apoptosisinducing activities in cancer cells

Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity

A phase I/II study of carfilzomib 2-10-min infusion in patients with advanced solid tumors

Phase I study of 30-minute infusion of carfilzomib as single agent or in combination with low-dose dexamethasone in patients with relapsed and/or refractory multiple myeloma

Alzheimer's disease progression in caenorhabditis elegans and neuronal cultures

Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer

Morphological and functional retinal impairment in Alzheimer's disease patients

Evidence of the neuroprotective role of citicoline in glaucoma patients

Enhancement of Retinal Function and of Neural Conduction Along the Visual Pathway Induced by Treatment with Citicoline Eye Drops in Liposomal Formulation in Open Angle Glaucoma: A Pilot Electrofunctional Study

Citicoline and Retinal Ganglion Cells: Effects on Morphology and Function

Next-generation proteasome inhibitors for cancer therapy

Structural defects in the regulatory particle-core particle interface of the proteasome induce a novel proteasome stress response

Reconfiguration of the proteasome during chaperone-mediated assembly

Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like activity of the proteasome

Functional retinal impairment in type 1 diabetic patients without any signs of retinopathy

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

Human metabolism of the proteasome inhibitor bortezomib: identification of circulating metabolites. Drug Metabolism and Disposition: The Biological Fate of Chemicals

Bortezomib-induced syndrome of inappropriate antidiuresis in a patient with multiple myeloma: A case report and literature review

Ubiquitin-mediated proteasome degradation regulates optic fissure fusion

Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains

CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation

Inhibition of constitutive NF-kappa B activation in mantle cell lymphoma B cells leads to induction of cell cycle arrest and apoptosis

Association between Early Neuroretinal Dysfunction and Peripheral Motor Unit Loss in Patients with Type 1 Diabetes Mellitus

Activation of retinal Müller cells in response to glucose variability

Mechanisms underlying ubiquitination

Differential roles of proteasome and immunoproteasome regulators Pa28αβ, Pa28γ and Pa200 in the degradation of oxidized proteins

THE IMMUNOPROTEASOME, THE 20S PROTEASOME, AND THE PA28αβ PROTEASOME REGULATOR ARE OXIDATIVE-STRESS-ADAPTIVE PROTEOLYTIC COMPLEXES

Oxidative Stress is the Principal Contributor to Inflammasome Activation in Retinal Pigment Epithelium Cells with Defunct Proteasomes and Autophagy

CEP-18770: A novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib

Vascular endothelial growth factor triggers signaling cascades mediating multiple myeloma cell growth and migration

Heterozygous Truncating Variants in POMP Escape Nonsense-Mediated Decay and Cause a Unique Immune Dysregulatory Syndrome

Marizomib, a proteasome inhibitor for all seasons: preclinical profile and a framework for clinical trials

Drug repurposing: progress, challenges and recommendations

Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis

Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis

/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37

Cellular processing of myocilin

Pharmacokinetics and safety of carfilzomib in patients with relapsed multiple myeloma and end-stage renal disease (ESRD): an open-label, single-arm, phase I study

Quality of Life in Glaucoma: A Review of the Literature

Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor

an important role during various stages of the coronavirus infection cycle

Diverse polyubiquitin interaction properties of ubiquitin-associated domains

Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases

The ubiquitin-proteasome system meets angiogenesis

Functionally different α-synuclein inclusions yield insight into Parkinson's disease pathology

New investigational drugs with single-agent activity in multiple myeloma

A mammalian nervous system-specific plasma membrane proteasome complex that modulates neuronal function

PACemakers of proteasome core particle assembly

Protein Kinase G Positively Regulates Proteasome-Mediated Degradation of Misfolded Proteins

p53 reactivation with induction of massive apoptosis-1 (PRIMA-1) inhibits amyloid aggregation of mutant p53 in cancer cells

Enhanced rate of degradation of basic proteins by 26S immunoproteasomes

PA28αβ reduces size and increases hydrophilicity of 20S immunoproteasome peptide products

Degradation of oxidized proteins by the proteasome: Distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways

Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors

Pharmacokinetic and pharmacodynamic study of two doses of bortezomib in patients with relapsed multiple myeloma

Resveratrol reduces amyloid-beta (Aβ₁₋₄₂)-induced paralysis through targeting proteostasis in an Alzheimer model of Caenorhabditis elegans

Comparative resistance of the 20S and 26S proteasome to oxidative stress

Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress

Glaucoma-associated myocilin: A better understanding but much more to learn

Management of treatment-emergent peripheral neuropathy in multiple myeloma

Ixazomib for the treatment of multiple myeloma

Extended follow-up of a phase II trial in relapsed, refractory multiple myeloma:: final time-to-event results from the SUMMIT trial

A phase 2 study of bortezomib in relapsed, refractory myeloma

Phase 1 study of twice-weekly ixazomib, an oral proteasome inhibitor, in relapsed/refractory multiple myeloma patients

Frequency, characteristics, and reversibility of peripheral neuropathy during treatment of advanced multiple myeloma with bortezomib

Twice-weekly ixazomib in combination with lenalidomide-dexamethasone in patients with newly diagnosed multiple myeloma

Twice-Weekly Oral MLN9708 (Ixazomib Citrate), An Investigational Proteasome Inhibitor

Dexamethasone (Dex) In Patients (Pts) With Newly Diagnosed Multiple Myeloma (MM): Final Phase 1 Results and Phase 2 Data

Pomalidomide, bortezomib, and dexamethasone for patients with relapsed or refractory multiple myeloma previously treated with lenalidomide (OPTIMISMM): a randomised, open-label

Bortezomib or high-dose dexamethasone for relapsed multiple myeloma

Extended follow-up of a phase 3 trial in relapsed multiple myeloma: final time-to-event results of the APEX trial

Lenalidomide, bortezomib, and dexamethasone combination therapy in patients with newly diagnosed multiple myeloma

A phase 2 trial of lenalidomide, bortezomib, and dexamethasone in patients with relapsed and relapsed/refractory myeloma

Bortezomib for the Treatment of Hematologic Malignancies: 15 Years Later

Bortezomib-Based Therapy for Newly Diagnosed Mantle-Cell Lymphoma

Association between bortezomib dose intensity and overall survival in mantle cell lymphoma patients on frontline VR-CAP

Frontline bortezomib, rituximab, cyclophosphamide, doxorubicin, and prednisone (VR-CAP) versus rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in transplantation-ineligible patients with newly diagnosed mantle cell lymphoma: final overall survival results of a randomised, open-label, phase 3 study

Steroid-induced glaucoma: Epidemiology, pathophysiology, and clinical management

Citicoline) in Glaucoma: Rationale of Its Use, Current Evidence and Future Perspectives

Chaperone-mediated pathway of proteasome regulatory particle assembly

Positioning of proteasome inhibitors in therapy of solid malignancies

Impaired Visual Search in Children with Rett Syndrome

Parkin reverses intracellular beta-amyloid accumulation and its negative effects on proteasome function

PRAD1, a candidate BCL1 oncogene: mapping and expression in centrocytic lymphoma

Degradation of ornithine decarboxylase in mammalian cells is ATP dependent but ubiquitin independent

Bortezomib, lenalidomide, and dexamethasone as induction therapy prior to autologous transplant in multiple myeloma

Superiority of bortezomib, thalidomide, and dexamethasone (VTD) as induction pretransplantation therapy in multiple myeloma: a randomized phase 3 PETHEMA/GEM study

An evolutionarily conserved pathway controls proteasome homeostasis

Regulation of proteasome assembly and activity in health and disease

Front-line transplantation program with lenalidomide, bortezomib, and dexamethasone combination as induction and consolidation followed by lenalidomide maintenance in patients with multiple myeloma: a phase II study by the Intergroupe Francophone du Myélome

Bortezomib plus CHOP-rituximab for previously untreated diffuse large B-cell lymphoma and mantle cell lymphoma

Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome

Kinetic mechanism of activation by cardiolipin (diphosphatidylglycerol) of the rat liver multicatalytic proteinase

The proteasome inhibitor NPI-0052 is a more effective inducer of apoptosis than bortezomib in lymphocytes from patients with chronic lymphocytic leukemia

The proteasome antechamber maintains substrates in an unfolded state

Christian de Duve: Explorer of the cell who discovered new organelles by using a centrifuge

Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening

Assembly and function of the proteasome

Ubiquitin-like proteins and Rpn10 play cooperative roles in ubiquitin-dependent proteolysis

Multiple Proteasome-Interacting Proteins Assist the Assembly of the Yeast 19S Regulatory Particle

Ubiquilin-1 overexpression increases the lifespan and delays accumulation of Huntingtin aggregates in the R6/2 mouse model of Huntington's disease

Shaping proteostasis at the cellular, tissue, and organismal level

Persistent overall survival benefit and no increased risk of second malignancies with bortezomib-melphalan-prednisone versus melphalan-prednisone in patients with previously untreated multiple myeloma

Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma

Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma

Tumor lysis syndrome associated with bortezomib: A post-hoc analysis after signal detection using the US Food and Drug Administration Adverse Event Reporting System. Anti-Cancer Drugs

CEP-18770 (delanzomib) in combination with dexamethasone and lenalidomide inhibits the growth of multiple myeloma

The proteasome inhibitor CEP-18770 enhances the anti-myeloma activity of bortezomib and melphalan

Antiangiogenic and anti-multiple myeloma effects of oprozomib (OPZ) alone and in combination with pomalidomide (Pom) and/or dexamethasone (Dex)

Success in translational research: lessons from the development of bortezomib

A phase 1/2 study of the oral proteasome inhibitor ixazomib in relapsed or refractory AL amyloidosis

A phase I/II dose-escalation study investigating all-oral ixazomibmelphalan-prednisone induction followed by single-agent ixazomib maintenance in transplantineligible newly diagnosed multiple myeloma

Panobinostat plus bortezomib and dexamethasone versus placebo plus bortezomib and dexamethasone in patients with relapsed or relapsed and refractory multiple myeloma: a multicentre, randomised, double-blind phase 3 trial

Panobinostat plus bortezomib and dexamethasone versus placebo plus bortezomib and dexamethasone in patients with relapsed or relapsed and refractory multiple myeloma: a multicentre, randomised, double-blind phase 3 trial

Overall survival of patients with relapsed multiple myeloma treated with panobinostat or placebo plus bortezomib and dexamethasone (the PANORAMA 1 trial): a randomised

Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from β-Amyloid Toxicity

Copper(II) ions affect the gating dynamics of the 20S proteasome: a molecular and in cell study

Cationic porphyrins are tunable gatekeepers of the 20S proteasome

Cationic Porphyrins Are Reversible Proteasome Inhibitors

Parkin truncating variants result in a loss-of-function phenotype

Proteasome biology is compromised in white matter after asphyxic cardiac arrest in neonatal piglets

PAC1 gene knockout reveals an essential role of chaperone-mediated 20S proteasome biogenesis and latent 20S proteasomes in cellular homeostasis

Thymoproteasomes produce unique peptide motifs for positive selection of CD8 + T cells

Human erythrocyte multicatalytic proteinase: activation and binding to sulfated galactoand lactosylceramides

Molecular and Structural Basis of the Proteasome α Subunit Assembly Mechanism Mediated by the Proteasome-Assembling Chaperone PAC3-PAC4 Heterodimer

A ratiometric naphthalimide sensor for live cell imaging of copper(i)

Nigral injection of a proteasomal inhibitor, lactacystin, induces widespread glial cell activation and shows various phenotypes of Parkinson's disease in young and adult mouse

Structural and functional evidence for citicoline binding and modulation of 20S proteasome activity: Novel insights into its pro-proteostatic effect

Retention of Mitochondria in Mature Human Red Blood Cells as the Result of

The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S

The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S

The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S

Defective proteasome biogenesis into skin fibroblasts isolated from Rett syndrome subjects with MeCP2 nonsense mutations

Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution

Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution

Diagnosing the decline in pharmaceutical R&D efficiency

Cilostazol, a phosphodiesterase 3 inhibitor, activates proteasome-mediated proteolysis and attenuates tauopathy and cognitive decline

Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade

Huntingtin-Encoded Polyglutamine Expansions Form Amyloid-like Protein Aggregates In Vitro and In Vivo

The Ubiquitin-Proteasome System in Huntington's Disease: Are Proteasomes Impaired, Initiators of Disease, or Coming to the Rescue?

Regulation of proteasome activity in health and disease

The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle

Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis

Severe acute respiratory syndrome coronavirus replication is severely impaired by MG132 due to proteasome-independent inhibition of M-calpain

The dynamic state of body constituents

Studies in Protein Metabolism Vii. the Metabolism of Tyrosine

The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design

Immunoproteasome deficiency protects in the retina after optic nerve crush

Autocatalytic processing of the 20S proteasome

Proteasome from Thermoplasma acidophilum: a threonine protease

O-GlcNAcylation Signal Mediates Proteasome Inhibitor Resistance in Cancer Cells by Stabilizing NRF1

Expression of mutated mouse myocilin induces open-angle glaucoma in transgenic mice

Molecular basis of differential sensitivity of myeloma cells to clinically relevant bolus treatment with bortezomib

The size of the proteasomal substrate determines whether its degradation will be mediated by mono-or polyubiquitylation

Transition metal complexes as proteasome inhibitors for cancer treatment

Efficacy and safety of carfilzomib in relapsed and/or refractory multiple myeloma: systematic review and meta-analysis of 14 trials

Oprozomib, pomalidomide, and Dexamethasone in Patients With Relapsed and/or Refractory Multiple Myeloma

Synthesis, Structure, and Anticancer Activity of Gallium(III) Complexes with Asymmetric Tridentate Ligands: Growth Inhibition and Apoptosis Induction of Cisplatin-Resistant Neuroblastoma Cells. Inorganic Chemistry

Telomerase inhibition and cell growth arrest by G-quadruplex interactive agent in multiple myeloma

Role of the ubiquitin-proteasome in protein quality control and signaling: implication in the pathogenesis of eye diseases

Insulin-degrading enzyme is activated by the C-terminus of α-synuclein

Insulin-degrading enzyme prevents α-synuclein fibril formation in a nonproteolytical manner

Structural Organization of the 19S Proteasome Lid: Insights from MS of Intact Complexes

Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination

The use of hollow mesoporous silica nanospheres to encapsulate bortezomib and improve efficacy for non-small cell lung cancer therapy

Mutations in the ubiquitinbinding domain of OPTN/optineurin interfere with autophagy-mediated degradation of misfolded proteins by a dominant-negative mechanism

Processing of optineurin in neuronal cells

Rod bipolar cells dysfunction occurs before ganglion cells loss in excitotoxin-damaged mouse retina

Proteasome Inhibitors: Harnessing Proteostasis to Combat Disease

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease

CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival

Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome

A phase 2 study of single-agent carfilzomib (PX-171-003-A1) in patients with relapsed and refractory multiple myeloma

Integrated safety profile of single-agent carfilzomib: experience from 526 patients enrolled in 4 phase II clinical studies

The role of the proteasome activator PA28 in MHC class I antigen processing

The role of the proteasome in the generation of MHC class I ligands and immune responses

The role of the proteasome in the generation of MHC class I ligands and immune responses

Of Mice and Men: Proteasome's Role in LPS-Induced Inflammation and Tolerance

The release of labeled amino acids from the proteins of rat liver slices

Total synthesis of the potent proteasome inhibitor epoxomicin: a useful tool for understanding proteasome biology

Pharmacodynamic and efficacy studies of the novel proteasome inhibitor NPI-0052 (marizomib) in a human plasmacytoma xenograft murine model

Effects of food on the clinical pharmacokinetics of anticancer agents: underlying mechanisms and implications for oral chemotherapy

Defects in autophagy caused by glaucoma-associated mutations in optineurin

Proteasome dysfunction in alveolar type 2 epithelial cells is associated with acute respiratory distress syndrome

Distinct Proteasome Subpopulations in the Alveolar Space of Patients with the Acute Respiratory Distress Syndrome

Alveolar extracellular 20S proteasome in patients with acute respiratory distress syndrome

O-GlcNAc signalling: implications for cancer cell biology

Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

Phase 1 study of ixazomib, an investigational proteasome inhibitor, in J o u r n a l P r e -p r o o f advanced non-hematologic malignancies

Reversal of long-term dendritic spine alterations in Alzheimer disease models

Could a Common Mechanism of Protein Degradation Impairment Underlie Many Neurodegenerative Diseases?

Could a Common Mechanism of Protein Degradation Impairment Underlie Many Neurodegenerative Diseases?

Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry

Docking of the Proteasomal ATPases' C-termini in the 20S Proteasomes alpha Ring Opens the Gate for Substrate Entry

ATP Binding to PAN or the 26S ATPases Causes Association with the 20S Proteasome, Gate Opening, and Translocation of Unfolded Proteins

Ixazomib: an investigational drug for the treatment of lymphoproliferative disorders

Targeting the proteostasis network in Huntington's disease

Proteasome Activation is Mediated via a Functional Switch of the Rpt6 C-terminal Tail Following Chaperone-dependent Assembly

Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae

Parkin promotes proteasomal degradation of p62: implication of selective vulnerability of neuronal cells in the pathogenesis of Parkinson's disease

Bortezomib-based versus nonbortezomib-based induction treatment before autologous stem-cell transplantation in patients with previously untreated multiple myeloma: a meta-analysis of phase III randomized, controlled trials

Sorting out the trash: the spatial nature of eukaryotic protein quality control

Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits

A phase 1 clinical trial evaluating marizomib, pomalidomide and low-dose dexamethasone in relapsed and refractory multiple myeloma (NPI-0052-107): final study results

Daratumumab plus bortezomib and dexamethasone versus bortezomib and dexamethasone in relapsed or refractory multiple myeloma: updated analysis of CASTOR

The Proteasome: More Than a Means to an End

VIEWPOINT An English I'ranslation of Alzheimer's 1907 Paper, " ijber eine eigenartige Erlranliung der Hirnrinde

Health-Related Quality-of-Life Results From the Open-Label, Randomized, Phase III ASPIRE Trial Evaluating Carfilzomib, Lenalidomide, and Dexamethasone Versus Lenalidomide and Dexamethasone in Patients With Relapsed Multiple Myeloma

Carfilzomib, lenalidomide, and dexamethasone for relapsed multiple

The role of ixazomib as an augmented conditioning therapy in salvage autologous stem cell transplant (ASCT) and as a post-ASCT consolidation and maintenance strategy in patients with relapsed multiple myeloma (ACCoRd [UK-MRA Myeloma XII] trial): study protocol for a Phase III randomised controlled trial

Histochemically-reactive zinc in amyloid plaques, angiopathy, and degenerating neurons of Alzheimer's diseased brains

Proteasome inhibition promotes mono-ubiquitination and nuclear translocation of mature (52 kDa) PINK1. Biochemical and Biophysical Research Communications

Novel proteasome inhibitor PS-341 inhibits activation of nuclear factorkappa B, cell survival, tumor growth, and angiogenesis in squamous cell carcinoma

Altered Functions and Interactions of Glaucoma-Associated Mutants of Optineurin

Protein misfolding in neurodegenerative diseases: implications and strategies

Proteasome inhibition potentiates antitumor effects of photodynamic therapy in mice through induction of endoplasmic reticulum stress and unfolded protein response

Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction

The synaptic accumulation of hyperphosphorylated tau oligomers in Alzheimer disease is associated with dysfunction of the ubiquitin-proteasome system

TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8+ T cells

Pathophysiology of diabetic retinopathy

Transgenic mice expressing caspase-6-derived N-terminal fragments of mutant huntingtin develop neurologic abnormalities with predominant cytoplasmic inclusion pathology composed largely of a smaller proteolytic derivative

Antiviral prophylaxis for varicella zoster virus infections in patients with myeloma in the era of novel therapies

The proteasome inhibitor PS-341 in cancer therapy

Efficacy of four different regimens in 64 mantle-cell lymphoma cases: clinicopathologic comparison with 498 other non-Hodgkin's lymphoma subtypes. European Organization for the Research and Treatment of Cancer Lymphoma Cooperative Group

A Practical Review of Proteasome Pharmacology

A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers

Proteomic analysis of protein phosphorylation and ubiquitination in Alzheimer's disease

An asymmetric interface between the regulatory and core particles of the proteasome

Investigational agent MLN9708/2238 targets tumor-suppressor miR33b in MM cells

alpha-synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome

Decreased proteasomal activity causes age-related phenotypes and promotes the development of metabolic abnormalities

New comprehensive studies of a gold(III) Dithiocarbamate complex with proven anticancer properties: Aqueous dissolution with cyclodextrins, pharmacokinetics and upstream inhibition of the ubiquitin-proteasome pathway

Incorporation of the Rpn12 Subunit Couples Completion of Proteasome Regulatory Particle Lid Assembly to Lid-Base Joining

The intrinsically disordered Sem1 protein functions as a molecular tether during proteasome lid biogenesis

Heterohexameric ring arrangement of the eukaryotic proteasomal ATPases: implications for proteasome structure and assembly

A Single α Helix Drives Extensive Remodeling of the Proteasome Lid and Completion of Regulatory Particle Assembly

Identification of aneuploidy-tolerating mutations

Effects of aneuploidy on cellular physiology and cell division in haploid yeast

Aneuploidy: cells losing their balance

A proteasome inhibitor prevents activation of NF-kappa B and stabilizes a newly phosphorylated form of I kappa B-alpha that is still bound to NF-kappa B

Pyrazolone Small Molecules Displaying Therapeutic Potential in Amyotrophic Lateral Sclerosis

Neuroprotective Effects of Betulin in Pharmacological and Transgenic Caenorhabditis elegans Models of Parkinson's Disease

Abeta inhibits the proteasome and enhances amyloid and tau accumulation

Porphyrin as Diagnostic and Therapeutic Agent

Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers

Insights into Proteasome Conformation Dynamics and Intersubunit Communication

Multiple functions of insulindegrading enzyme: a metabolic crosslight?

On the Horizon: Targeting Next-Generation Immune Checkpoints for Cancer Treatment

Characterization of the testis-specific proteasome subunit α4s in mammals

Induction of the Immunoproteasome Subunit Lmp7 Links Proteostasis and Immunity in α-Synuclein Aggregation Disorders. EBioMedicine

Parkin directly modulates 26S proteasome activity

Parkin ubiquitinates and promotes the degradation of RanBP2

The structure of the mammalian 20S proteasome at 2.75 A resolution

PA200, a nuclear proteasome activator involved in DNA repair

Purification and assay of proteasome activator PA200

Relative contributions of the five major human cytochromes P450, 1A2, 2C9, 2C19, 2D6, and 3A4, to the hepatic metabolism of the proteasome inhibitor bortezomib. Drug Metabolism and Disposition: The Biological Fate of Chemicals

Chaperoning the Cancer: The Proteostatic Functions of the Heat Shock Proteins in Cancer

Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress

Cancer and dementia: Two sides of the same coin?

The dynamics of early-state transcriptional changes and J o u r n a l P r e -p r o o f aggregate formation in a Huntington's disease cell model

Nuclear factor-kappaB in development, prevention, and therapy of cancer

Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism

The early history of the ubiquitin field

Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast

Effect of the CYP3A inhibitor ketoconazole on the pharmacokinetics and pharmacodynamics of bortezomib in patients with advanced solid tumors: a prospective, multicenter, open-label, randomized, two-way crossover drug-drug interaction study

Cyclophosphamide, bortezomib, and dexamethasone therapy in AL amyloidosis is associated with high clonal response rates and prolonged progression-free survival

Inactivating PSMB5 mutations and P-glycoprotein (multidrug resistance-associated protein/ATP-binding cassette B1) mediate resistance to proteasome inhibitors: ex vivo efficacy of (immuno)proteasome inhibitors in mononuclear blood cells from patients with rheumatoid arthritis

Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome

2D-and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study

Regulating protein breakdown through proteasome phosphorylation

Exploring the regulation of proteasome function by subunit phosphorylation

26S Proteasomes are rapidly activated by diverse hormones and physiological states that raise cAMP and cause Rpn6 phosphorylation

On the Trails of the Proteasome Fold: Structural and Functional Analysis of the Ancestral β-Subunit Protein Anbu

Hemin Inhibits Ubiquitin-Dependent Proteolysis in both a Higher Plant and Yeast

Linkage between the proteasome pathway and neurodegenerative diseases and aging

An open-label, single-arm, phase 2 (PX-171-004) study of single-agent carfilzomib in bortezomib-naive patients with relapsed and/or refractory multiple myeloma

Structure of ubiquitin refined at 1.8 A resolution

Amyloid beta -peptide inhibition of the PKA/CREB pathway and long-term potentiation: reversibility by drugs that enhance cAMP signaling

Cancer genome landscapes

Phase I/II study of the novel proteasome inhibitor delanzomib (CEP-18770) for relapsed and refractory multiple myeloma

Ocular antigen does not cause disease unless presented in the context of inflammation

Mantle cell lymphoma: 2017 update on diagnosis, risk-stratification, and clinical management

VEGF release by retinal glia depends on both oxygen and glucose supply

The Lewy body in Parkinson's disease: molecules implicated in the formation and degradation of alpha-synuclein aggregates

The Lewy body in Parkinson's disease and related neurodegenerative disorders

Physiological function of myocilin and its role in the pathogenesis of glaucoma in the trabecular meshwork (Review)

Müller Cell-Derived VEGF Is Essential for Diabetes-Induced Retinal Inflammation and Vascular Leakage

Phase 2 dose-expansion study (PX-171-006) of carfilzomib, lenalidomide, and low-dose dexamethasone in relapsed or progressive multiple myeloma

Interactions of SARS coronavirus nucleocapsid protein with the host cell proteasome subunit p42

The proteasome-interacting Ecm29 protein disassembles the 26S proteasome in response to oxidative stress

Regulation of the 26S proteasome complex during oxidative stress

Clinical pharmacokinetics, metabolism, and drug-drug interaction of carfilzomib. Drug Metabolism and Disposition: The Biological Fate of Chemicals

In Vitro Metabolism of Oprozomib, an Oral Proteasome Inhibitor: Role of Epoxide Hydrolases and Cytochrome P450s

Carfilzomib-Associated Cardiovascular Adverse Events: A Systematic Review and Meta-analysis

Does aneuploidy cause cancer?

A novel regimen combining high dose cytarabine and bortezomib has activity in multiply relapsed and refractory mantle cell lymphoma -long-term results of a multicenter observation study

The Pathophysiology and Treatment of Glaucoma

Mantle cell lymphoma. A clinicopathologic study of 68 cases from the Nebraska Lymphoma Study Group

Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

How we treat glioblastoma

Structurally-defined deubiquitinase inhibitors provide opportunities to investigate disease mechanisms

Phase 2 study of carfilzomib, thalidomide, and low-dose dexamethasone as induction and consolidation in newly diagnosed, transplant eligible patients with multiple myeloma, the carthadex trial

Synthetic peptide-based activators of the proteasome

Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme

Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex

The discovery of ubiquitin-dependent proteolysis

Aneuploidy -Cancer's Fatal Flaw?

Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology

Crystal structure of a low molecular weight activator Blm-pep with yeast 20S proteasome -insights into the enzyme activation mechanism

Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasomes

Proteasome activator (PA28) subunits, alpha, beta and gamma (Ki antigen

Accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor 3

An AAA Motor-Driven Mechanical Switch in Rpn11 Controls Deubiquitination at the 26S Proteasome

Structure of the Rpn11-Rpn8 dimer reveals mechanisms of substrate deubiquitination during proteasomal degradation

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

PAC1-PAC2 proteasome assembly chaperone retains the core α4-α7 assembly intermediates in the cytoplasm

Increased plasma levels of 20S proteasome alpha-subunit in glaucoma patients: an observational pilot study

Inhibition of USP14 enhances the sensitivity of breast cancer to enzalutamide

Thymoproteasome subunit-β5T generates peptide-MHC complexes specialized for positive selection

Proteasome Activators, PA28α and PA28β, Govern Development of Microvascular Injury in Diabetic Nephropathy and Retinopathy

Aggregated Myocilin Induces Russell Bodies and Causes Apoptosis

Irreversible potent activation and reversible inhibition of trypsin-like activity of 20S proteasome purified from Xenopus oocytes by fatty acid

Hsp90-mediated assembly of the 26 S proteasome is involved in major histocompatibility complex class I antigen processing

Isostructurality Among 5 Solvatomorphs of Betulin: X-Ray Structure and Characterization

Crosstalk between the proteasome system and autophagy in the clearance of α-synuclein

Natural Compounds with Proteasome Inhibitory Activity for Cancer Prevention and Treatment

Pharmacokinetics, pharmacodynamics, metabolism, distribution, and excretion of carfilzomib in rats. Drug Metabolism and Disposition: The Biological Fate of Chemicals

Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling

Inhibiting autophagy reduces retinal degeneration caused by protein misfolding

A cryptic protease couples deubiquitination and degradation by the proteasome

Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1

N-Terminal Ubiquitination of Amyloidogenic Proteins Triggers Removal of Their Oligomers by the Proteasome Holoenzyme

A dual inhibitor of the proteasome catalytic subunits LMP2 and Y attenuates disease progression in mouse models of Alzheimer's disease

Induction of autophagy in rats upon overexpression of wild-type and mutant optineurin gene

The ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry

The deubiquitinating enzyme ataxin-3 does not modulate disease progression in a knock-in mouse model of Huntington disease

O -GlcNAc Modification Is an Endogenous Inhibitor of the Proteasome

Structural insights into the regulatory particle of the proteasome from Methanocaldococcus jannaschii

Proteasome function is regulated by cyclic AMP-dependent protein kinase through phosphorylation of Rpt6

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors

MnFe2O4 nanoparticles accelerate the clearance of mutant huntingtin selectively through ubiquitin-proteasome system

l-Tryptophan Schiff base cadmium(II) complexes as a new class of proteasome inhibitors and apoptosis inducers in human breast cancer cells

Redox-Active Metal Complexes for Anticancer Therapy

Inhibition of tumor proteasome activity by gold-dithiocarbamato complexes via both redoxdependent and -independent processes

Suppresses Tumor Cell Proliferation by Acting as an Endogenous Proteasome Inhibitor via Targeting the Proteasome Assembly Factor POMP

Coagulopathy and Antiphospholipid Antibodies in Patients with SARS-Cov-2

Organic cadmium complexes as proteasome inhibitors and apoptosis inducers in human breast cancer cells

Cefepime, a fourth-generation cephalosporin, in complex with manganese, inhibits proteasome activity and induces the apoptosis of human breast cancer cells

A novel nickel complex works as a proteasomal deubiquitinase inhibitor for cancer therapy

FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells

mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy

A phase I/II study of bortezomib in combination with paclitaxel, carboplatin, and concurrent thoracic radiation therapy for non-small-cell lung cancer: North Central Cancer Treatment Group (NCCTG)-N0321

Deletion of the huntingtin polyglutamine stretch enhances neuronal autophagy and longevity in mice

Clarifying the molecular mechanism associated with carfilzomib resistance in human multiple myeloma using microarray gene expression profile and genetic interaction network

Design and Synthesis of an Orally Bioavailable and Selective Peptide Epoxyketone Proteasome Inhibitor (PR-047)

Transgenic mice expressing the Tyr437His mutant of human myocilin protein develop glaucoma

An oral second-generation proteasome inhibitor oprozomib significantly inhibits lung cancer in a p53 independent manner in vitro

MG132 enhances the radiosensitivity of lung cancer cells in vitro and in vivo

The proteasome maturation protein POMP increases proteasome assembly and activity in psoriatic lesional skin

Proteasome impairment by α-synuclein

Critical elements in proteasome assembly

The authors woulk like to thank Prof. G. Manni for his fruitful discussion. This work was supported by the Italian Ministery of University and Research (PRIN

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