key: cord-0832426-d31sy49k
authors: Schoeps, Benjamin; Eckfeld, Celina; Flüter, Laura; Keppler, Selina; Mishra, Ritu; Knolle, Percy; Bayerl, Felix; Böttcher, Jan; Hermann, Chris D.; Häußler, Daniel; Krüger, Achim
title: Identification of invariant chain CD74 as a functional receptor of tissue inhibitor of metalloproteinases-1 (TIMP-1)
date: 2021-08-12
journal: J Biol Chem
DOI: 10.1016/j.jbc.2021.101072
sha: ba7c80f88a7d9c77d5779b906f3e3456600f7670
doc_id: 832426
cord_uid: d31sy49k

Multifunctionality of tissue inhibitor of metalloproteinases-1 (TIMP-1) comprising antiproteolytic as well as cytokinic activity has been attributed to its N-terminal and C-terminal domains, respectively. The molecular basis of the emerging proinflammatory cytokinic activity of TIMP-1 is still not completely understood. The cytokine receptor invariant chain (CD74) is involved in many inflammation-associated diseases and is highly expressed by immune cells. CD74 triggers zeta chain–associated protein kinase-70 (ZAP-70) signaling–associated activation upon interaction with its only known ligand, the macrophage migration inhibitory factor. Here, we demonstrate TIMP-1–CD74 interaction by coimmunoprecipitation and confocal microscopy in cells engineered to overexpress CD74. In silico docking in HADDOCK predicted regions of the N-terminal domain of TIMP-1 (N-TIMP-1) to interact with CD74. This was experimentally confirmed by confocal microscopy demonstrating that recombinant N-TIMP-1 lacking the entire C-terminal domain was sufficient to bind CD74. Interaction of TIMP-1 with endogenously expressed CD74 was demonstrated in the Namalwa B lymphoma cell line by dot blot binding assays as well as confocal microscopy. Functionally, we demonstrated that TIMP-1–CD74 interaction triggered intracellular ZAP-70 activation. N-TIMP-1 was sufficient to induce ZAP-70 activation and interference with the cytokine-binding site of CD74 using a synthetic peptide–abrogated TIMP-1-mediated ZAP-70 activation. Altogether, we here identified CD74 as a receptor and mediator of cytokinic TIMP-1 activity and revealed TIMP-1 as moonlighting protein harboring both cytokinic and antiproteolytic activity within its N-terminal domain. Recognition of this functional TIMP-1–CD74 interaction may shed new light on clinical attempts to therapeutically target ligand-induced CD74 activity in cancer and other inflammatory diseases.

Multifunctionality of tissue inhibitor of metalloproteinases-1 (TIMP-1) comprising antiproteolytic as well as cytokinic activity has been attributed to its N-terminal and C-terminal domains, respectively. The molecular basis of the emerging proinflammatory cytokinic activity of TIMP-1 is still not completely understood. The cytokine receptor invariant chain (CD74) is involved in many inflammation-associated diseases and is highly expressed by immune cells. CD74 triggers zeta chainassociated protein kinase-70 (ZAP-70) signaling-associated activation upon interaction with its only known ligand, the macrophage migration inhibitory factor. Here, we demonstrate TIMP-1-CD74 interaction by coimmunoprecipitation and confocal microscopy in cells engineered to overexpress CD74. In silico docking in HADDOCK predicted regions of the Nterminal domain of TIMP-1 (N-TIMP-1) to interact with CD74. This was experimentally confirmed by confocal microscopy demonstrating that recombinant N-TIMP-1 lacking the entire C-terminal domain was sufficient to bind CD74. Interaction of TIMP-1 with endogenously expressed CD74 was demonstrated in the Namalwa B lymphoma cell line by dot blot binding assays as well as confocal microscopy. Functionally, we demonstrated that TIMP-1-CD74 interaction triggered intracellular ZAP-70 activation. N-TIMP-1 was sufficient to induce ZAP-70 activation and interference with the cytokine-binding site of CD74 using a synthetic peptide-abrogated TIMP-1-mediated ZAP-70 activation. Altogether, we here identified CD74 as a receptor and mediator of cytokinic TIMP-1 activity and revealed TIMP-1 as moonlighting protein harboring both cytokinic and antiproteolytic activity within its N-terminal domain. Recognition of this functional TIMP-1-CD74 interaction may shed new light on clinical attempts to therapeutically target ligandinduced CD74 activity in cancer and other inflammatory diseases.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a soluble metalloproteinase inhibitor, which is found at high concentrations in the circulation of patients suffering from inflammation-associated diseases (1) (2) (3) . Originally described as the first natural collagenase inhibitor (4) , it was revealed that TIMP-1 is identical to erythroid-potentiating activity (5), a potent growth factor for erythroid progenitors. Subsequent studies revealed further effects of TIMP-1 on cells of the hematopoietic system, including B lymphoma cells (6) (7) (8) . Interestingly, many of the observed TIMP-1-mediated effects were shown to be independent of its antiproteolytic activity (6, 9) , which so far was the only major function exhibited by the N-terminal domain (10) . Several years later, it was discovered that C-terminal domain-mediated interaction of TIMP-1 with the tetraspanin CD63 (11, 12) induces proteolysis-independent cytokinic signaling resulting in a variety of protumorigenic, prometastatic, and proinflammatory effects (13) (14) (15) (16) . Hence, the multifunctionality of TIMP-1 was attributed to the existence of two physically distinct domains, the N-terminal domain mediating antiproteolytic activity as well as the C-terminal domain mediating signaling activity via CD63 (17) .

In an abstract for a symposium, without provision of experimental data (18) , it was suggested that invariant chain (CD74) may be a possible interaction partner of TIMP-1. CD74 is, like TIMP-1, associated with inflammatory diseases (19) and is discussed as a therapeutic target in cancer (20) and autoimmune diseases (21) and notably also in the ongoing SARS-CoV-2 pandemic (22) . CD74 plays an essential role in many biological processes (23), including antigen presentation (24) and B cell development (25) . Homotrimers of CD74 bind major histocompatibility complex (MHC) class II complexes to assist their assembly and subcellular localization (23, 26) . In addition, MHC-free CD74 can also be found on the surface of immune cells (23, 27) , where it serves as the receptor for the cytokine macrophage migration inhibitory factor (MIF) (28) . Functionally, the MIF-CD74 interaction was shown to induce intracellular zeta chainassociated protein kinase-70 (ZAP-70) signaling in B lymphoma cells (29) . In the present study, we identified CD74 as a functionally active receptor for TIMP-1, mediating cytokinic signaling activity of TIMP-1. Furthermore, we identified the antiproteolytic N-terminal domain of TIMP-1 to be a functional cytokinic interaction site as well.

To investigate a possible TIMP-1-CD74-interaction, we overexpressed CD74 in the human LX-2 cell line, which resulted in highly increased RNA (Fig. 1A) , total protein ( Fig. 1B) , as well as surface protein (Fig. 1C ) levels of CD74. Physical interaction between TIMP-1 and CD74 could be demonstrated by coimmunoprecipitation using the crude lysate of CD74-overexpressing cells (Fig. 1D) . Furthermore, colocalization of fluorescently labeled TIMP-1 with CD74 was observed (Fig. 1E) . Altogether, these data show that TIMP-1 binds to CD74.

In silico prediction of TIMP-1-CD74 interacting residues by molecular docking

To shed light on possible interaction sites involved in TIMP-1-CD74 binding, we performed in silico molecular docking using the Haddock algorithm (30) , which predicts the most likely conformation of two interacting molecules ( Fig. 2A) . For this, we used the full-length crystal structure of TIMP-1 (PDB ID: 1uea) (31) and the trimerized extracellular domain of CD74 (PDB ID: 1iie) (32). Of 10,000 randomly assigned interactions between TIMP-1 and CD74, the 200 energetically most favorable interactions were further refined by semiflexible annealing. The structures of these refined TIMP-1-CD74 complexes were compared and complexes with similar orientation were clustered. Of the resulting 22 clusters, the three, in respect to energetic considerations, bestranked clusters (clusters 2, 8, and 13 (Table S1)) were further analyzed.

To gain more insight in the binding sites that are involved in TIMP-1-CD74 interaction, we visualized a representative complex structure for each cluster (complex 2, complex 8, and complex 13) (Fig. 2B ) and used the SpotOn algorithm, which identifies and classifies interfacial residues of protein-protein interactions (33, 34) . This analysis revealed that, in complexes 8 and 13, TIMP-1 would interact with CD74 mainly via its N-terminal domain (Fig. 2B) , including the wedge-shaped ridge at the N terminus of TIMP-1 ( Fig. 2C and Fig. S1A ). This interaction was predicted to involve all three monomers of the CD74 homotrimer ( Fig. 2D and Fig. S1B ). In contrast, complex 2 suggested binding to CD74 only via the C-terminal domain of TIMP-1 (Fig. 2 , B and C and Fig. S1A) .

Interestingly, some of the suggested residues on CD74 that participate in the interaction with TIMP-1 (highlighted in Fig. S1B ) were previously also predicted to be involved in binding of the MIF to CD74 (35) .

In vitro validation of the in silico-predicted docking site of TIMP-1 with CD74

The in silico analysis suggested two possible binding orientations of TIMP-1 to CD74. First, binding to CD74 would be mediated via the N-terminal domain of TIMP-1 (complex 8 and complex 13). Second, the interaction with CD74 would be mediated via the C-terminal domain of TIMP-1 (complex 2).

To experimentally validate one of the proposed binding mechanisms, we deleted the C-terminal domain of TIMP-1 and used the purified N-terminal domain (N-TIMP-1) for in vitro studies. Confocal microscopy revealed that N-TIMP-1, similar to full-length TIMP-1 (Fig. 1B) , colocalized with CD74 (Fig. 3A) . This experimental approach demonstrates that the N-terminal domain of TIMP-1 is sufficient to bind to CD74, supporting the binding mechanism suggested by complexes 8 and 13.

As a proof of concept, we aimed to investigate whether the identified TIMP-1-CD74 interaction also occurs in cells with natural expression of CD74. Analysis of published RNA-Seq data derived from the human protein atlas (36) revealed that cells in the immune cell compartment, particularly B cells, highly expressed CD74 (Fig. S2) . Therefore, we used the Namalwa human B lymphoma cell line, which highly expressed CD74 (Fig. S3A) , for further studies on TIMP-1-CD74 interactions. Dot blot binding assays using cell lysates of Namalwa cells revealed, in comparison with the negative control BSA, markedly increased binding of CD74 to immobilized recombinant TIMP-1 to a similar extent as the positive control recombinant MIF (Fig. 3B ). This increase was totally abolished when TIMP-1-coated dots were incubated with lysates from CD74 knockdown cells ( Fig. 3B and Fig. S3 , B and C). Furthermore, colocalization of fluorescently labeled TIMP-1 and CD74 in intact Namalwa cells was observed using confocal microscopy (Fig. 3C) . Importantly, N-TIMP-1 was sufficient to bind to Namalwa-derived CD74 (Fig. 3D ).

Next, we examined whether the identified TIMP-1-CD74 interaction is functional. For this, we analyzed phosphorylation of ZAP-70, a known downstream effector of CD74 signaling in B lymphoma cells upon binding of MIF (29) . Stimulation of Namalwa cells with recombinant TIMP-1 triggered ZAP-70 phosphorylation (Fig. 4A ). Importantly, TIMP-1-mediated ZAP-70 phosphorylation was dependent on CD74 because shRNAi-mediated knockdown of CD74 ( Fig. 4B ) completely abolished this effect (Fig. 4C ). Of note, GAPDH and total ZAP-70 levels were not affected by TIMP-1 stimulation (Fig. S4 ). To further analyze the relevance of the above-identified interfacial residues of TIMP-1-CD74 interaction, we stimulated Namalwa cells with N-TIMP-1 and observed that the N-terminal domain was sufficient to trigger ZAP-70 phosphorylation (Fig. 4D ). The abovementioned complexes 8 and 13, which were identified to reflect the most probable binding mechanism, predicted a TIMP-1-binding region on CD74 that is similar to the previously published binding region of MIF on CD74 (35) . Because MIF-CD74 interaction can be blocked with the synthetic CD74-binding peptide C36L1 occupying this binding region on CD74 (37), we used C36L1 to test for the plausibility of the in silico-revealed TIMP-1-interaction regions on CD74. Indeed, preincubation of cells with this peptide abolished TIMP-1-CD74-mediated ZAP-70 phosphorylation (Fig. 4E) , indicating that the known cytokinebinding site on CD74 is involved in TIMP-1-CD74 In silico prediction of TIMP-1-CD74 interacting residues by molecular docking. A, illustration of the molecular docking approach to predict the interaction site of TIMP-1 (PDB ID: 1uea) with CD74 (PDB ID: 1ile). Known crystal structures are shown in surface depiction, and dashed lines indicate unresolved structural regions. B, representative complexes of the three energetically most favorable TIMP-1-CD74 clusters obtained from the haddock docking approach and illustrated with UCSF Chimera. The N-terminal domain of TIMP-1 is shown in light blue, the C-terminal domain is shown in blue, and the CTC motif of TIMP-1 is shown in orange. Monomer 1 of the CD74 homotrimer is shown in dark green, monomer 2 is shown in light green, and monomer 3 is shown in yellow. The indicated score represents the respective calculated Haddock score, an indicator of the intermolecular energy of the respective complexes. C, surface representation of TIMP-1 with interface regions participating in the interaction with CD74 highlighted in red. The CTC motif is highlighted in orange (top). TIMP-1 molecules were turned around the x, y, and z axes as indicated. D, surface representation of CD74 with interface regions participating in the interaction with TIMP-1 highlighted in red. CD74 molecules were turned around the x, y, and z axes as depicted. interaction. Of note, we could exclude effects of the peptide on cell viability during the time frame of the experiment (Fig. S5 ).

In this study, we identified CD74 as the functional receptor of TIMP-1. Interaction of TIMP-1 with CD74 was observed in lysates derived from cells of different origins, namely CD74-overexpressing hepatic stellate cells as well as Namalwa B lymphoma cells with natural expression of CD74. This finding was further supported by confocal microscopy revealing that fluorescently labeled exogenous TIMP-1 binds to the receptor CD74. The observation that not all TIMP-1 molecules colocalized with CD74 in cells points at additional TIMP-1-binding proteins. Indeed, several cell surface proteins are known to bind TIMP-1, including CD63 (11), LRP-1 (38, 39) , and ADAM10 (40) .

Further molecular analyses on the identified interaction between TIMP-1 and CD74 led to the observation that TIMP-1 interacts with CD74 via its N-terminal domain because N-TIMP-1 alone was sufficient to bind to CD74 as well as trigger ZAP-70 activation. Still, molecular docking analyses indicated that some amino acids located in the C-terminal domain facing the wedge-shaped ridge of TIMP-1 (41) possibly participate in the interaction with CD74 as well. Therefore, it is possible that these residues can be involved in modulation of the TIMP-1-CD74 interaction. Such contribution of amino acids of the C-terminal domain to the binding of TIMP-1 to other known N-terminal interaction partners has already been described in the context of its antiproteolytic activity because residues in the C-terminal domain are not decisive for, but involved in, fine-tuning of TIMP-1-metalloproteinase interactions (42, 43) .

TIMP-1 is a multifunctional protein with versatile impact on cellular processes. Its functions result from its two-domain structure harboring metalloproteinase inhibitory as well as cytokine activities (17) . So far, the N-terminal domain of TIMP-1 was almost entirely recognized to be responsible for the antiproteolytic function (10), while it emerged that the C-terminal domain is the mediator of its cytokinic activity (11, 12) . This appreciation now has to be revised as we here demonstrate that the N-terminal domain of TIMP-1 binds to CD74 and triggers ZAP-70 activation in Namalwa B lymphoma cells. Thus, the multifunctionality of TIMP-1 expands beyond functional separation of two distinct domains because the N-terminal domain harbors both metalloproteinase inhibitory and cytokinic functions.

CD74 is, like TIMP-1, highly expressed in inflammatory conditions (19) and has been shown to be involved in a multitude of biological processes (23), including antigen presentation (24) and cellular signaling (29) . So far, the only known ligand of CD74 was the proinflammatory cytokine MIF, and MIF-CD74 interaction was shown to induce ZAP-70 signaling in B lymphoma cells (29) . The clinical relevance of ligand-induced CD74 activity is demonstrated by clinical trials using anti-CD74 antibodies for the therapy of malignant B cell lymphoma (44) as well as studies aiming at interference with CD74-MIF signaling in the ongoing SARS-CoV-2 pandemic (22) . Our finding that ZAP-70 signaling is also triggered upon interaction of TIMP-1 with CD74 suggests that TIMP-1 and MIF may exert similar cellular responses. Interestingly, the molecular modeling analyses suggested that TIMP-1 binds to the known MIF-binding site on CD74 (35, 37) . This was experimentally validated by interference with TIMP-1-CD74 signaling using a peptide known to block MIF-CD74 interaction (37) . The physiological consequence of TIMP-1-CD74 interactions, including a potential competition between TIMP-1 and MIF in binding to CD74, warrants further investigation.

In conclusion, our findings render TIMP-1 as a moonlighting protein (45) , where the N-terminal domain harbors both the well-known antiproteolytic activity and a so far unknown signaling function.

The human LX-2 cell line was cultured in DMEM (Biochrom AG) supplemented with 2% fetal bovine serum, 1% glutamine, and 2% penicillin/streptomycin at 37 C and 5% CO 2 . The human B lymphoma cell line Namalwa was cultured in RPMI 1640 (Thermo Fisher Scientific Inc) supplemented with 7.5% fetal bovine serum.

Experimental procedures were followed as described previously (46) . Briefly, 5 × 10 6 293T cells were seeded in 10-cm dishes. After 1 day, media were replaced and cotransfection of transfer plasmids purchased from Sigma Aldrich (shCD74 #1 (TRCN0000008636, 96.65% knockdown, validated), shCD74 #2 (TRCN0000008635, 98.35% knockdown, validated), shCD74 #3 (TRCN0000008639, 74.14% knockdown, validated), (for knockdown efficiencies, see Fig. 4B and Fig. S3 , B and C)) with lentiviral packaging plasmids (pMD.GP, pRSVrev, pMD.G) was performed using Lipofectamine 2000 (cat.#11668019, Thermo Fisher Scientific Inc). shRNA that does not target any known human or mouse gene (shNT) was used as a control. Two days later, lentiviral particles were harvested from the supernatant and frozen at −80 C. Target cells were infected with lentiviral particles in presence of 8 μg/ ml Polybrene (cat.#TR-1003-G, Sigma Aldrich), and selection of transduced cells was performed using 10 μg/ml puromycin. the nucleus is shown in blue. The scale bars represent 2 μm. D, representative dot blot assay investigating N-TIMP-1 binding to CD74. Immobilized N-TIMP-1 was incubated with nondenatured cell lysate from B lymphoma cells. Binding of cell lysate-derived CD74 to uncoated (n = 5) and N-TIMP-1-coated dots (n = 5) was determined by immunoblotting against CD74 (left panel) and quantified using densitometric analysis of dots (right panel). B and D, results are represented as the mean ± SD. For statistical analyses, a one-sample t test was used in case of normal distribution or a one-sample Wilcoxon test in absence of normal distribution, *p ≤ 0.05. MIF, macrophage migration inhibitory factor; N-TIMP-1, N-terminal domain of TIMP-1; TIMP-1, tissue inhibitor of metalloproteinases-1. 

CD74-overexpressing LX-2 cells were generated using a lentiviral vector (plasmid pHIV7) containing a spleen focusforming promoter for overexpression of the CD74 isoform p33. The DNA sequence of CD74 was synthesized by Invitrogen GeneArt Gene Synthesis (Thermo Fisher Scientific Inc). Control cells were generated using an empty vector.

Recombinant human (N-)TIMP-1 was expressed in an endotoxin-free mammalian cell culture system of HEK293F cells (Thermo Fisher Scientific) and purified in a three-step protocol using an Äktapure Fast Protein Liquid Chromatography system (Cytiva) as described previously (16) . For stimulation of cells for subsequent analysis of intracellular signaling, cells were incubated in serum-free media containing 0.1% BSA for 24 h before stimulation. After serum starvation, cells were exposed to pathophysiological concentrations of TIMP-1 (500 ng/ml) (14) , N-TIMP-1 (392 ng/ml, equimolar to TIMP-1), or MIF (100 ng/ml, Abcam, cat. #ab75432) for 10 min and whole-cell extracts were isolated using RIPA lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.25% SDS, 1% NP-40, 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Carl Roth). Concentrations of isolated proteins were determined using the Pierce BCA Protein Assay Reagents (Thermo Fisher Scientific Inc). For interference with TIMP-1-CD74-mediated signaling, cells were preincubated with the CD74-blocking peptide C36L1 (37) (200 μM, KSSQSVFYSSNNKNYLA-NH2, peptides & elephants GmbH) for 6 h before stimulation.

For coimmunoprecipitation experiments, nondenatured cell lysates of TIMP-1-stimulated (500 ng/ml) CD74overexpressing LX-2 cells were prepared by a nondenaturing cell lysis buffer (20 mM Tris HCl, pH 8, 137 mM NaCl, 1% NP-40, 2 mM EDTA, including phosphatase and proteinase inhibitors). Cell lysates were incubated with agarose beads conjugated with anti-CD74 (cat.# sc-6262-AC, Santa Cruz Biotechnology) or IgG antibodies (cat.# sc-2343-AC, Santa Cruz Biotechnology). Before incubation with cell lysates, conjugated beads were blocked for 1 h at room temperature (RT) with 5% BSA in PBS to avoid unspecific binding. Beads were then centrifuged (1000g, 30 s, 4 C) and washed three times with PBS. The remaining cell pellet was resuspended in the nondenatured cell lysate of CD74-overexpressing LX-2 cells and incubated overnight at 4 C while rotating. After centrifugation (1000g, 30 s, 4 C) and washing (3× with PBS), the remaining cell pellet containing the beads and bead-bound molecules were resuspended in the loading buffer (0.2 M Tris, 0.03% SDS, 0.07% glycerin, 0.002% bromophenol blue, 0.05% β-mercaptoethanol) and boiled at 95 C for 5 min to elute bound molecules from the beads. Eluates were then subjected to Western blot analysis to detect protein abundance of CD74 and TIMP-1.

For Western blot analyses, equal amounts of protein were resuspended in the loading buffer (0.2 M Tris, 0.03% SDS, 0.07% glycerin, 0.002% bromophenol blue, 0.05% β-mercaptoethanol) and incubated for 5 min at 95 C. Samples were centrifuged (13,000g, 3 min, 4 C) and separated by a 10% or 12.5% SDS PAGE. Proteins were transferred onto a nitrocellulose membrane (Amersham Bioscience) using a Trans-Blot semidry blotting system (Bio-Rad Laboratories Inc). After protein transfer, membranes were blocked with 5% BSA in TBS-T (1× TBS containing 0.1% Tween 20) for 1 h at RT and incubated with primary antibodies against P-ZAP-70 (Tyr319, cat. #2701S, Cell Signaling Technology), human CD74 (1:1000, # sc-20082, Santa Cruz Biotechnology), or GAPDH (cat. #sc-25778, Santa Cruz Biotechnology) diluted in 5% BSA in TBS-T overnight at 4 C. Membranes were washed three times with TBS-T at RT and incubated with secondary goat anti-rabbit IgG HRP-conjugated antibody solutions (1:5000; cat. #31462, Thermo Fisher Scientific Inc) for 1 h at RT. Membranes were washed three times with TBS-T, and bands were visualized using the Pierce ECL substrate (Thermo Fisher Scientific Inc). Bands were detected and quantified using the ChemiDoc imaging system (Bio-Rad Laboratories Inc) and the ImageLab Software (Bio-Rad Laboratories Inc).

Binding of CD74 to TIMP-1, N-TIMP-1, and MIF was analyzed by chemiluminescent Dot blot assays. Human recombinant TIMP-1 (6.7 μg), N-TIMP-1 (equimolar concentrated), and MIF (equimolar concentrated), diluted in TBS buffer, were immobilized on a nitrocellulose membrane using a Bio-Dot microfiltration apparatus (Bio-Rad Laboratories Inc) according to the manufacturer's instructions. Uncoated dots were incubated with TBS buffer only. All dots were blocked with 5% BSA in TBS-T and incubated overnight at 4 C with nondenatured cell lysates derived from the B lymphoma cell line Namalwa (20 μg/ml). Cell lysis was performed using a nondenaturing cell lysis buffer (20 mM Tris HCl, pH 8.0, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA). Membranes were washed twice with TBS-T, incubated with primary antibodies against human CD74 (1:1000, # sc-20082, Santa Cruz Biotechnology) until the antibody solution was drained through the wells completely. Membranes were washed twice with TBS-T and incubated with secondary goat anti-rabbit IgG HRP-conjugated antibody solutions (1:5000; cat. #31462, Thermo Fisher Scientific Inc). After washing with TBS-T, dots were visualized using the ChemiDoc imaging system (Bio-Rad stimulated with 500 ng/ml TIMP-1 after preincubation with or without an anti-CD74 blocking peptide. A, C, and D, results are represented as the mean ± SD. For statistical analyses, a one-sample t test was used in case of normal distribution or a one-sample Wilcoxon test in the absence of normal distribution, *p ≤ 0.05; n.s., not significant. MIF, macrophage migration inhibitory factor; N-TIMP-1, N-terminal domainof TIMP-1; TIMP-1, tissue inhibitor of metalloproteinases-1; ZAP-70, zeta chain-associated protein kinase-70.

Laboratories Inc). Dot intensity was quantified using the ImageLab Software (Bio-Rad Laboratories Inc).

One hundred microgram of recombinant human TIMP-1 was fluorescently labeled using the Alexa Fluor 555 Microscale Protein Labeling Kit (cat. #A30007, Thermo Fisher Inc) according to manufacturer's instructions. For stimulation of cells with fluorescently labeled TIMP-1 or N-TIMP-1, 6 × 10 5 cells were stimulated with Alexa 555 TIMP-1 (70 nM) or Alexa 555 N-TIMP-1 (equimolar concentrated) for 10 min on ice and the nonbound ligand was rigorously washed off with ice-cold PBS (3×). Cells were fixed by 4% PFA for 15 min at RT, washed twice with PBS, and permeabilized by 0.5% Triton X-100 in PBS for 10 min at RT. After washing (2× with PBS), cells were blocked with 5% horse serum in PBS and stained for CD74 using anti-CD74 antibodies (1:500, #ab108393, Abcam) diluted in the blocking solution overnight at 4 C. After washing (2× with PBS), cells were incubated with the secondary antibody (1:500, cat. #SA5-10038, donkey anti-rabbit IgG-DyLight488, Thermo Fisher Scientific Inc) diluted in the blocking solution for 1 h at RT. Nuclei were stained with DAPI (1:5000, #A4099, AppliChem) for 10 min at RT, mounted using ROTI-Mount FluorCare (Carl Roth) and stored at 4 C until imaging. Imaging was performed with an inverted Leica TCS SP8 confocal microscope with White Light Laser and HyD photodetectors using a HC PL APO CS2 64×/OIL objective. Analysis of pictures and 3D surface models of nuclei and colocalization regions were performed using Imaris 9.3.1 (Oxford Instruments).

Viability of Namalwa cells treated with or without the CD74 blocking peptide C36L1 (37) (200 μM, KSSQSVFYSSNNKNYLA-NH2, peptides & elephants GmbH) was monitored by flow cytometry. Cells were starved for 24 h before stimulation in serum-free media containing 0.1% BSA. After 6 h of treatment with or without the blocking peptide, cells were transferred to 96-well v-bottom plates (TPP Techno Plastic Products AG), and dead cells were stained using 7-AAD (1:40, cat.#420403, BioLegend). Staining of cell surface CD74 was performed by incubation of cells with FITCconjugated anti-CD74 antibodies (1:100, cat.# 11-0748-42, BioLegend) for 30 min at 4 C and subsequent staining of dead cells using 7-AAD (1:40, cat.#420403, BioLegend). Flow cytometry was performed with the SA3800 Spectral Analyzer (Sony Biotechnology Inc) and analyzed using FlowJo Software (version.10.6., Tree Star, Inc).

For in silico prediction of TIMP-1-CD74 complexes, the molecular docking software Haddock (30) (semiflexible docking) was used. The spatial coordinates for the molecular docking were retrieved from the Protein Data Bank for a 20-kDa human TIMP-1 monomer (31) (PDB ID: 1uea) and the trimerized extracellular domain of the 33-kDa isoform of human CD74 (32) (PDB ID: 1lle). For docking, the coordinates were entered as whole molecules. The coordinates for both molecules were entered in the CPORT server (47) to generate active and passive residues that are necessary for the molecular docking with Haddock. Coordinates for TIMP-1 and CD74 as whole molecules as well as the respective active and passive residues were entered in the Haddock webserver. This docking approach generated 400 complexes of TIMP-1 and CD74. 186 of these complexes clustered in 22 clusters. The three bestranked structures (according to the energetically most favorable conformation) were utilized for further analysis and were visualized.

The three clusters with the lowest haddock score (balanced mode) were utilized for interface analysis and visualization. For interface analysis, the SpotOn webserver (33, 34) was used. The respective complex coordinates were entered as whole complexes in the SpotOn software that calculated interface residues. Comparison of the interface residues on TIMP-1 and CD74 of the different complexes was conducted by Microsoft Excel. For representation of complexes, UCSF Chimera (version 1.15) was used.

Statistical analysis was performed using the statistical software SPSS, version 24.0. Normal distribution was tested by Shapiro-Wilk tests. Groups were compared using Student's t test for independent samples in the case of normal distribution or the nonparametric Mann-Whitney test for independent variables in the absence of normal distribution. For Western blot densitometric analyses, a one-sample t test compared with the reference value 1 was used in case of normal distribution or a one-sample Wilcoxon test compared with the reference value 1 in the absence of normal distribution.

The data supporting the findings of this study are contained within the article.

Supporting information-This article contains supporting information (35, 36) .

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Blockade of MIF-CD74 signalling on macrophages and dendritic cells restores the antitumour immune response against metastatic melanoma

Lowdensity lipoprotein receptor-related protein-1 mediates endocytic clearance of tissue inhibitor of metalloproteinases-1 and promotes its cytokine-like activities

Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons

The isolated N-terminal domains of TIMP-1 and TIMP-3 are insufficient for ADAM10 inhibition

Tissue inhibitors of metalloproteinases

Directed evolution of the metalloproteinase inhibitor TIMP-1 reveals that its N-and C-terminal domains cooperate in matrix metalloproteinase recognition

Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E. coli

Phase I study of the anti-CD74 monoclonal antibody milatuzumab (hLL1) in patients with previously treated B-cell lymphomas

Moonlighting proteins: An intriguing mode of multitasking

Tissue inhibitor of metalloproteinases (TIMP)-1 creates a premetastatic niche in the liver through SDF-1/CXCR4-dependent neutrophil recruitment in mice

CPORT: A consensus interface predictor and its performance in prediction-driven docking with HADDOCK

Acknowledgments-The B cell lymphoma cell line was kindly provided by Ghislain Opdenakker (KU Leuven, Leuven, Belgium). Molecular graphics were generated with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, USA, with support from NIH P41-GM103311.