key: cord-0281708-gwt9jbze authors: Karthigeyan, Krithika P.; Zhang, Lizhi; Loiselle, David R.; Haystead, Timothy A. J.; Bhat, Menakshi; Yount, Jacob S.; Kwiek, Jesse J. title: A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation date: 2021-05-07 journal: bioRxiv DOI: 10.1101/2021.05.07.443132 sha: 6df04a65a40d808bb11d618ccbc0f9e5eb0e5e79 doc_id: 281708 cord_uid: gwt9jbze Cells acquire fatty acids from dietary sources or via de novo palmitate production by fatty acid synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers and during replication of many viruses. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo fatty acid synthesis contributes to host or viral protein acylation has been traditionally difficult to study. We describe a cell permeable, click-chemistry compatible alkynyl-acetate analog (Alk-4) that functions as a reporter of FASN-dependent protein acylation. In a FASN-dependent manner, Alk-4 selectively labeled the cellular protein interferon-induced transmembrane protein 3 (IFITM3) at its palmitoylation sites, and the HIV-1 matrix protein at its myristoylation site. Alk-4 metabolic labeling also enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states. Long chain fatty acids (FA) are essential components of lipid bilayers, are used to store energy 31 liberated by β-oxidation, and are covalently attached to proteins to increase hydrophobicity and 32 regulate subcellular localization. 1 Long chain fatty acids can be obtained exogenously through 33 dietary sources, or endogenously via de novo fatty acid biosynthesis. 2 Mammalian fatty acid 34 synthase (FASN) is a 272 kDa cytosolic enzyme that catalyzes the complete de novo 35 identify acylated proteins. 14 While protein myristoylation site prediction is facilitated by a 48 consensus sequence motif on nearly all myristoylated proteins (Met-Gly-XXX-Ser/Thr), 1 protein 49 palmitoylation site prediction remains challenging due to the lack of a consensus sequence. 15 To measure acyl-group synthesis mediated by FASN and the fate of the de novo synthesized 51 fatty acids, one must use 14 C labeled acetate, which suffers from low detection sensitivity, 52 general complications associated with radioisotope work, 16 and an inability to selectively enrich 53 acylated proteins. Over the last decade, bioorthogonal labeling and detection of protein fatty 54 acylation using click chemistry compatible analogs of palmitate and myristate have provided 55 quick and sensitive methods for detection of protein acylations. 17, 18 The copper-catalyzed 56 azide-alkyne cycloaddition (CuAAC) reactions enable labeling of cells with alkynyl analogs of 57 fatty acids that can be reacted with azides conjugated to suitable detection tags, such as 58 fluorophores, or affinity tags, including biotin. 19,20 Although very useful, palmitate and myristate 59 analogs only measure the acylation state of proteins modified by the exogenous chemical 60 V Labeling of IFITM3 by Alk-4 requires FASN. We have previously shown that IFITM3 108 palmitoylation is required for its antiviral activity against influenza virus infection. 22, 33 To 109 determine if FASN-mediated de novo fatty acid biosynthesis contributes to an IFNβ-regulated 110 IFITM3-mediated antiviral response, we measured endogenous IFITM3 labeling by Alk-4 in 111 wild-type (WT) and FASN knockout HAP1 cells. As expected, IFNβ induced endogenous 112 IFITM3 expression, and IFITM3 upregulation was independent of FASN expression ( Figure 113 2e). In WT cells, Alk-4 treatment resulted in robust endogenous IFITM3 labeling that was 114 absent in FASN-deficient cells (Figure 2f ). Thus, we show for the first time that FASN 115 contributes to the palmitoylation of endogenous IFITM3. Owing to the observations that IFITM3 116 is required for an effective IFNβ-mediated anti-influenza response, 32 that palmitoylation of 117 IFITM3 is required for its antiviral activity 32 , and that FASN regulates Alk-4 mediated IFITM3 118 palmitoylation (Figure 2f ), we sought to determine the effect of FASN expression on IFNβ-119 mediated inhibition of influenza virus infection. In the absence of IFNβ, FASN expression had 120 no effect on influenza infection ( Figure 2g ). However, IFNβ-mediated inhibition of influenza 121 virus infection was significantly decreased in the absence of FASN expression (Figure 2g) , 122 suggesting that FASN-dependent palmitate synthesis likely contributes to the palmitoylation-123 dependent antiviral activity of IFITM3. 124 Alk-4 labeling of myristoylated proteins is FASN dependent. Acetyl CoA is condensed with 125 malonyl-CoA and elongated by FASN to generate palmitate for protein palmitoylation. To 126 generate myristoyl CoA for myristoylation, palmitoyl CoA must be β-oxidized to myristoyl CoA 127 before it is covalently attached to glycine residues by N-myristoyl transferases (Figure 4d ). 34,1 128 To determine if Alk-4 is metabolized into a fatty acid analog that can selectively label 129 myristoylated proteins, we tested whether a known myristoylated protein, HIV-1 matrix protein 130 We describe a click-chemistry compatible FASN-substrate, Alk-4 (5-hexynoate), which 177 selectively labels both palmitoylated (e.g. IFITM3, CD9) and myristoylated (e.g. HIV-1 matrix) 178 proteins. Click chemistry compatible substrate analogs like Alk-4 overcome several inherent 179 disadvantages of radiolabeling, such as long sample processing and film exposure times with 180 low sensitivity. 16 Moreover, click chemistry reactions can be combined with several detection 181 methods that are compatible with high throughput applications including mass spectrometry-182 based proteomics, flow cytometry, fluorescence microscopy, and live cell imaging. 21 Beyond 183 identification of FASN-dependent protein acylation, Alk-4 has more functionality than a 184 radiolabeled FASN substrate because it can be reacted with azido-biotin to facilitate 185 streptavidin-based purification of FASN-dependent acylated proteins. We are not the first to 186 suggest the utility of Alk-4, which has previously been evaluated as a chemical tool to monitor 187 protein acetylation at shorter timescales, although it was noted that some of the acetylation Proteomics analysis of SA pulldown fraction revealed that 77% of proteins selectively 397 recovered in the WT Alk-4 cells are found in at least one palmitoyl proteome or experimentally 398 validated to be palmitoylated based on the SwissPalm database (Dataset 3). Additionally, 17% 399 of proteins are predicted to be palmitoylated, based both on GPS-lipid analysis using high 400 confidence settings, CSS-Palm, and SwissPalm (Dataset 1). GPS-Lipid analysis also revealed 401 3% of proteins are predicted to be myristoylated (consensus N-terminal Glycine and non-402 consensus sequence), while 2% of the proteins were not hits on prediction algorithms but had 403 isoforms and cysteines as per SwissPalm analysis. 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