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• W3157486080 abstract "Mammalian cells acquire fatty acids (FAs) 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 of the breast, prostate, and liver, among others, and is required during the replication of many viruses, such as dengue virus, hepatitis C, HIV-1, hepatitis B, and severe acute respiratory syndrome coronavirus 2, among others. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo FA synthesis contributes to host or viral protein acylation has been traditionally difficult to study. Here, we describe a cell-permeable and click chemistry–compatible alkynyl acetate analog (alkynyl acetic acid or 5-hexynoic acid [Alk-4]) that functions as a reporter of FASN-dependent protein acylation. In an FASN-dependent manner, Alk-4 selectively labels the cellular protein interferon-induced transmembrane protein 3 at its known palmitoylation sites, a process that is essential for the antiviral activity of the protein, and the HIV-1 matrix protein at its known myristoylation site, a process that is required for membrane targeting and particle assembly. 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. Mammalian cells acquire fatty acids (FAs) 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 of the breast, prostate, and liver, among others, and is required during the replication of many viruses, such as dengue virus, hepatitis C, HIV-1, hepatitis B, and severe acute respiratory syndrome coronavirus 2, among others. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo FA synthesis contributes to host or viral protein acylation has been traditionally difficult to study. Here, we describe a cell-permeable and click chemistry–compatible alkynyl acetate analog (alkynyl acetic acid or 5-hexynoic acid [Alk-4]) that functions as a reporter of FASN-dependent protein acylation. In an FASN-dependent manner, Alk-4 selectively labels the cellular protein interferon-induced transmembrane protein 3 at its known palmitoylation sites, a process that is essential for the antiviral activity of the protein, and the HIV-1 matrix protein at its known myristoylation site, a process that is required for membrane targeting and particle assembly. 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 (FAs) are essential components of lipid bilayers, are used to store energy liberated by β-oxidation, and are covalently attached to proteins to increase hydrophobicity and regulate subcellular localization (1Resh M.D. Fatty acylation of proteins: The long and the short of it.Prog. Lipid Res. 2016; 63: 120-131Crossref PubMed Scopus (141) Google Scholar). In mammalian cells, long-chain FAs can be obtained exogenously through dietary sources or endogenously via de novo FA biosynthesis (2Suburu J. Gu Z. Chen H. Chen W. Zhang H. Chen Y.Q. Fatty acid metabolism: Implications for diet, genetic variation, and disease.Food Biosci. 2013; 4: 1-12Crossref PubMed Scopus (18) Google Scholar). Mammalian fatty acid synthase (FASN) is a 272 kDa cytosolic enzyme that catalyzes the complete de novo synthesis of palmitate from acetyl-CoA and malonyl-CoA. The final product, palmitic acid (16:0), is then released from the thioesterase domain of FASN and can then be metabolized by β-oxidation into myristic acid in the mitochondria (14:0), elongated to other long-chain FA such as stearic acid, or elongated and desaturated into oleic acid in the endoplasmic reticulum (3Liu H. Liu J.Y. Wu X. Zhang J.T. Biochemistry, molecular biology, and pharmacology of fatty acid synthase, an emerging therapeutic target and diagnosis/prognosis marker.Int. J. Biochem. Mol. Biol. 2010; 1: 69-89PubMed Google Scholar). This process is aided by acyl-CoA synthetases, which activate the free FAs produced by FASN to their coenzyme-A linked thioesters. FAs synthesized de novo or acquired through dietary sources can be covalently attached to proteins by acyltransferases such as palmitoyl transferases and N-myristoyl transferases in a process called fatty acylation (1Resh M.D. Fatty acylation of proteins: The long and the short of it.Prog. Lipid Res. 2016; 63: 120-131Crossref PubMed Scopus (141) Google Scholar). FASN expression is highly regulated in cells, and its expression can change dramatically in response to stresses, such as starvation, lactation, or pathological states (3Liu H. Liu J.Y. Wu X. Zhang J.T. Biochemistry, molecular biology, and pharmacology of fatty acid synthase, an emerging therapeutic target and diagnosis/prognosis marker.Int. J. Biochem. Mol. Biol. 2010; 1: 69-89PubMed Google Scholar). Increased de novo FA biosynthesis and FASN upregulation have been observed in breast cancer, melanoma, and hepatocellular carcinoma (4Kuhajda F.P. Fatty-acid synthase and human cancer: New perspectives on its role in tumor biology.Nutrition. 2000; 16: 202-208Crossref PubMed Scopus (647) Google Scholar). Studies of enveloped viruses including hepatitis B virus (5Zhang H. Li H. Yang Y. Li S. Ren H. Zhang D. Hu H. Differential regulation of host genes including hepatic fatty acid synthase in HBV-transgenic mice.J. Proteome Res. 2013; 12: 2967-2979Crossref PubMed Scopus (14) Google Scholar), dengue virus (6Tongluan N. Ramphan S. Wintachai P. Jaresitthikunchai J. Khongwichit S. Wikan N. Rajakam S. Yoksan S. Wongsiriroj N. Roytrakul S. Smith D.R. Involvement of fatty acid synthase in dengue virus infection.Virol. J. 2017; 14: 28Crossref PubMed Scopus (26) Google Scholar), Epstein–Barr virus (7Li Y. Webster-Cyriaque J. Tomlinson C.C. Yohe M. Kenney S. Fatty acid synthase expression is induced by the Epstein-Barr virus immediate-early protein BRLF1 and is required for lytic viral gene expression.J. Virol. 2004; 78: 4197-4206Crossref PubMed Scopus (50) Google Scholar), hepatitis C virus (8Nasheri N. Joyce M. Rouleau Y. Yang P. Yao S. Tyrrell D.L. Pezacki J.P. Modulation of fatty acid synthase enzyme activity and expression during hepatitis C virus replication.Chem. Biol. 2013; 20: 570-582Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), HIV-1 (9Kulkarni M.M. Ratcliff A.N. Bhat M. Alwarawrah Y. Hughes P. Arcos J. Loiselle D. Torrelles J.B. Funderburg N.T. Haystead T.A. Kwiek J.J. Cellular fatty acid synthase is required for late stages of HIV-1 replication.Retrovirology. 2017; 14: 45Crossref PubMed Scopus (21) Google Scholar), chikungunya virus (10Bakhache W. Neyret A. McKellar J. Clop C. Bernard E. Weger-Lucarelli J. Briant L. Fatty acid synthase and stearoyl-CoA desaturase-1 are conserved druggable cofactors of Old World Alphavirus genome replication.Antiviral Res. 2019; 172: 104642Crossref PubMed Scopus (9) Google Scholar, 11Zhang N. Zhao H. Zhang L. Fatty acid synthase promotes the palmitoylation of Chikungunya virus nsP1.J. Virol. 2019; 93e01747-18Crossref PubMed Scopus (31) Google Scholar), and West Nile virus (12Krishnan M.N. Ng A. Sukumaran B. Gilfoy F.D. Uchil P.D. Sultana H. Brass A.L. Adametz R. Tsui M. Qian F. Montgomery R.R. Lev S. Mason P.W. Koski R.A. Elledge S.J. et al.RNA interference screen for human genes associated with West Nile virus infection.Nature. 2008; 455: 242-245Crossref PubMed Scopus (420) Google Scholar, 13Martín-Acebes M.A. Blázquez A.B. Jiménez de Oya N. Escribano-Romero E. Saiz J.C. West Nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids.PLoS One. 2011; 6e24970Crossref PubMed Scopus (108) Google Scholar) indicate that many viruses both upregulate and require host FASN activity for effective replication. The contributions of de novo–synthesized FA to post-translational modifications of viral and host proteins remain understudied. Identification of protein acylation has been challenging because of the lack of antibodies against lipid modifications and inefficiencies of standard mass spectrometry techniques to identify acylated proteins (14Hang H.C. Linder M.E. Exploring protein lipidation with chemical biology.Chem. Rev. 2011; 111: 6341-6358Crossref PubMed Scopus (84) Google Scholar). While protein myristoylation site prediction is facilitated by a consensus sequence motif on nearly all myristoylated proteins (Met-Gly-XXX-Ser/Thr) (1Resh M.D. Fatty acylation of proteins: The long and the short of it.Prog. Lipid Res. 2016; 63: 120-131Crossref PubMed Scopus (141) Google Scholar), protein palmitoylation site prediction remains challenging because of the lack of a consensus sequence (15Rodenburg R.N.P. Snijder J. Van De Waterbeemd M. Schouten A. Granneman J. Heck A.J.R. Gros P. Stochastic palmitoylation of accessible cysteines in membrane proteins revealed by native mass spectrometry.Nat. Commun. 2017; 8: 1280Crossref PubMed Scopus (33) Google Scholar). Measuring acyl-group synthesis mediated by FASN and the fate of the de novo–synthesized FAs using 3H labeled acetate suffers from low detection sensitivity, general complications associated with radioisotope work (16Draper J.M. Smith C.D. Palmitoyl acyltransferase assays and inhibitors (review).Mol. Membr. Biol. 2009; 26: 5-13Crossref PubMed Scopus (52) Google Scholar), and an inability to selectively enrich acylated proteins. Over the last decade, bioorthogonal labeling and detection of protein fatty acylation using click chemistry–compatible analogs of palmitate and myristate have provided quick and sensitive methods for detection of protein acylations (17Gao X. Hannoush R.N. A decade of click chemistry in protein palmitoylation: Impact on discovery and new biology.Cell Chem. Biol. 2018; 25: 236-246Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 18Yap M.C. Kostiuk M.A. Martin D.D.O. Perinpanayagam M.A. Hak P.G. Siddam A. Majjigapu J.R. Rajaiah G. Keller B.O. Prescher J.A. Wu P. Bertozzi C.R. Falck J.R. Berthiaume L.G. Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry.J. Lipid Res. 2010; 51: 1566-1580Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The copper-catalyzed azide–alkyne cycloaddition reactions enable labeling of cells with alkynyl analogs of FAs that can be reacted with azides conjugated to suitable detection tags, such as fluorophores, or affinity tags, including biotin (19Thiele C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. Shevchenko A. Kuerschner L. Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (77) Google Scholar, 20Ourailidou M.E. Zwinderman M.R.H. Dekker F.J. Bioorthogonal metabolic labelling with acyl-CoA reporters: Targeting protein acylation.MedChemComm. 2016; https://doi.org/10.1039/c5md00446bCrossref Scopus (5) Google Scholar). Although very useful, palmitate and myristate analogs only measure the acylation state of proteins modified by the exogenous chemical reporters. Given the critical role of FASN-dependent de novo–synthesized FAs in cancer, metabolic disorders, and viral replication, we posit that a bioorthogonal reporter of FASN-dependent protein acylation will facilitate a better understanding of the contributions of FASN-dependent protein fatty acylation to protein function, protein localization, and FASN-mediated pathogenesis. Here, we demonstrate the utility of alkynyl acetic acid or 5-hexynoic acid (Alk-4), a cell-permeable and click chemistry compound that labels proteins acylated by products of FASN-mediated de novo FA biosynthesis. Bioorthogonal reporters such as alkynyl palmitate (alkynyl palmitic acid or 15-hexadecynoic acid [Alk-16]) and alkynyl myristic acid or 13-tetradecynoic acid (Alk-12) are substrates of palmitoyltransferase and myristoyltransferase activity that are often used to identify palmitoylated and myristoylated proteins (21Charron G. Zhang M.M. Yount J.S. Wilson J. Raghavan A.S. Shamir E. Hang H.C. Robust fluorescent detection of protein fatty-acylation with chemical reporters.J. Am. Chem. Soc. 2009; 131: 4967-4975Crossref PubMed Scopus (219) Google Scholar, 22Yount J.S. Moltedo B. Yang Y.Y. Charron G. Moran T.M. López C.B. Hang H.C. Palmitoylome profiling reveals S-palmitoylation-dependent antiviral activity of IFITM3.Nat. Chem. Biol. 2010; 6: 610-614Crossref PubMed Scopus (258) Google Scholar, 23Chesarino N.M. Hach J.C. Chen J.L. Zaro B.W. Rajaram M.V. Turner J. Schlesinger L.S. Pratt M.R. Hang H.C. Yount J.S. Chemoproteomics reveals toll-like receptor fatty acylation.BMC Biol. 2014; 12: 91Crossref PubMed Scopus (39) Google Scholar). Alk-12 and Alk-16 are exogenous FA analogs that mimic the end product of FASN activity (palmitate) or the β-oxidation product of palmitate (myristate) and thus cannot be used to determine if de novo–synthesized FAs can be incorporated onto protein acylation sites. We hypothesized that a cell-permeable and bioorthogonal mimic of a putative FASN substrate, 5-hexynoate (termed Alk-4 here) (24Yang Y.Y. Ascano J.M. Hang H.C. Bioorthogonal chemical reporters for monitoring protein acetylation.J. Am. Chem. Soc. 2010; 132: 3640-3641Crossref PubMed Scopus (123) Google Scholar), could be used to study the contributions of FASN-mediated de novo FA synthesis to protein acylation (Fig. 1, A and B). To determine whether Alk-4 selectively labels palmitoylated proteins, we tested whether a known palmitoylated protein, interferon-induced transmembrane protein 3 (IFITM3), was labeled upon a 24-h treatment of cells with alkynyl acetate analogs of different carbon chain lengths, 4-pentynoic acid (Alk-3) and Alk-4, in comparison with the well-established palmitoylation reporter Alk-16 (Fig. 1, A and B). Alk-16 robustly labeled IFITM3 as detected by click chemistry tagging of immunoprecipitated IFITM3 with azidorhodamine and fluorescence gel scanning. Alk-4 also successfully labeled IFITM3, whereas Alk-3 showed minimal labeling (Fig. 2A). Next, we tested whether Alk-4 labeling of IFITM3 occurred on its known palmitoylated cysteines (22Yount J.S. Moltedo B. Yang Y.Y. Charron G. Moran T.M. López C.B. Hang H.C. Palmitoylome profiling reveals S-palmitoylation-dependent antiviral activity of IFITM3.Nat. Chem. Biol. 2010; 6: 610-614Crossref PubMed Scopus (258) Google Scholar). A triple cysteine to alanine palmitoylation-deficient mutant of IFITM3 (termed PalmΔ) was not labeled by either Alk-16 or Alk-4 (Fig. 2B). Hydroxylamine treatment is known to cleave the palmitoyl–thioester linkage (25Roth A.F. Wan J. Green W.N. Yates 3rd, J.R. Davis N.G. Proteomic identification of palmitoylated proteins.Methods. 2006; 40: 135Crossref PubMed Scopus (33) Google Scholar), and hydroxylamine treatment removed labeling of IFITM3 by Alk-4 (Fig. 2C). To further test the ability of Alk-4 to label palmitoylated proteins, we examined whether the tetraspanin CD9, which has six palmitoylated cysteines, was also labeled by Alk-4. Similar to IFITM3, CD9 was labeled by Alk-4, whereas a mutant CD9 in which its palmitoylated cysteines were mutated to alanine (termed CD9-PalmΔ) was not labeled (Fig. 2D). These results indicate that Alk-4 is metabolized into a click chemistry–functionalized FA adduct that is specifically incorporated onto protein palmitoylation sites.Figure 2FASN-dependent incorporation of Alk-4 at known protein palmitoylation sites. A, immunoprecipitated HA-IFITM3 from HA-IFITM3 transfected 293Ts followed by rhodamine azide click reaction revealed detectable labeling by Alk-16 and Alk-4 and minimal labeling by Alk-3. B, a triple cysteine to alanine IFITM3 mutant (PalmΔ) was not labeled by Alk-4, suggesting that Alk-4 labeling of IFITM3 occurred on known palmitoylated cysteines. C, Alk-4 labeling of IFITM3 is removed by hydroxylamine treatment, which removes palmitate groups from S-palmitoylated proteins. D, Alk-4 labeled CD9, whereas a mutant where its six palmitoylated cysteines were mutated to alanine was not labeled, revealing that Alk-4 labels CD9 on known palmitoylated cysteines (∗ indicates 25 kDa fluorescent molecular weight standard bleed through). E, DHHC palmitoyltransferase overexpression increased Alk-4 labeling of IFITM3, whereas a dominant negative mutant partially decreased labeling, suggesting that Alk-4 is metabolized into a long-chain fatty acid utilized by DHHC palmitoyltransferases. F, to test the requirement of FASN for labeling of endogenous IFITM3 in cells treated with IFNβ, HAP1 WT and FASN KO cells were labeled with Alk-4. Western blotting was done to confirm FASN levels in WT and KO cells and expression of endogenous IFITM3 on IFNβ treatment. G, Alk-4 labeling of endogenous IFITM3 was only observed in WT cells and not detected in FASN KO cells, indicating that FASN contributes to palmitoylation of IFITM3. H, IFNβ was significantly less effective at inhibiting influenza virus strain H1N1 infection in FASN KO cells (∗p = 0.0002), indicating that FASN is required for mounting of an effective IFNβ immune response against influenza virus, possibly through provision for fatty acyl groups for activation of IFITM3. DHHC, aspartate–histidine–histidine–cysteine; FASN, fatty acid synthase; HA, hemagglutinin; IFITM3, interferon-induced transmembrane protein 3; IFNβ, interferon beta.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Many proteins are reversibly palmitoylated at cysteine residues (26Mitchell D.A. Vasudevan A. Linder M.E. Deschenes R.J. Protein palmitoylation by a family of DHHC protein S-acyltransferases.J. Lipid Res. 2006; 47: 1118-1127Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) by aspartate–histidine–histidine–cysteine (DHHC) palmitoyltransferases. DHHC palmitoyltransferases primarily use palmitoyl-CoA (C16:0) to modify cysteine residues on proteins, although DHHCs can tolerate substrates with carbon chain lengths as short as 14 and as long as 20 (27Greaves J. Munro K.R. Davidson S.C. Riviere M. Wojno J. Smith T.K. Tomkinson N.C. Chamberlain L.H. Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E1365-E1374Crossref PubMed Scopus (69) Google Scholar, 28Jennings B.C. Linder M.E. DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities.J. Biol. Chem. 2012; 287: 7236-7245Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Acyl chains with fewer than 14 carbons have not been detected on cysteines, indicating that DHHC enzymes disfavor short-chain FAs as substrates (29Muszbek L. Haramura G. Cluette-Brown J.E. Van Cott E.M. Laposata M. The pool of fatty acids covalently bound to platelet proteins by thioester linkages can be altered by exogenously supplied fatty acids.Lipids. 1999; 34 Suppl: S331-S337Crossref PubMed Google Scholar, 30Hallak H. Muszbek L. Laposata M. Belmonte E. Brass L.F. Manning D.R. Covalent binding of arachidonate to G protein alpha subunits of human platelets.J. Biol. Chem. 1994; 269: 4713-4716Abstract Full Text PDF PubMed Google Scholar, 31Veit M. Reverey H. Schmidt M.F. Cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins.Biochem. J. 1996; 318: 163-172Crossref PubMed Scopus (49) Google Scholar, 32Thinon E. Fernandez J.P. Molina H. Hang H.C. Selective enrichment and direct analysis of protein S-palmitoylation sites.J. Proteome Res. 2018; 17: 1907-1922Crossref PubMed Scopus (26) Google Scholar). Given the selectivity of the DHHC palmitoyltransferases for long-chain FAs, we sought to determine whether labeling of IFITM3 by Alk-4 was affected by DHHC7 overexpression, which was previously shown to be among the enzymes that can catalyze IFITM3 palmitoylation (33McMichael T.M. Zhang L. Chemudupati M. Hach J.C. Kenney A.D. Hang H.C. Yount J.S. The palmitoyltransferase ZDHHC20 enhances interferon-induced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity.J. Biol. Chem. 2017; 292: 21517-21526Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In cells incubated with Alk-4, DHHC7 overexpression increased IFITM3 labeling, whereas overexpression of a dominant negative DHHC7 mutant decreased IFITM3 labeling (Fig. 2E). These results indicate that Alk-4 is metabolized into a long-chain FA that can be used as a substrate by DHHC palmitoyltransferases for protein palmitoylation. We have previously shown that IFITM3 palmitoylation is required for its antiviral activity against influenza virus infection (22Yount J.S. Moltedo B. Yang Y.Y. Charron G. Moran T.M. López C.B. Hang H.C. Palmitoylome profiling reveals S-palmitoylation-dependent antiviral activity of IFITM3.Nat. Chem. Biol. 2010; 6: 610-614Crossref PubMed Scopus (258) Google Scholar, 34Percher A. Ramakrishnan S. Thinon E. Yuan X. Yount J.S. Hang H.C. Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 4302-4307Crossref PubMed Scopus (77) Google Scholar). To determine if FASN-mediated de novo FA biosynthesis contributes to an interferon beta (IFNβ)–regulated IFITM3-mediated antiviral response, we measured endogenous IFITM3 labeling by Alk-4 in WT and FASN KO HAP1 cells. As expected, IFNβ induced endogenous IFITM3 expression, and IFITM3 upregulation was independent of FASN expression (Fig. 2F). In WT cells, Alk-4 treatment resulted in robust endogenous IFITM3 labeling that was absent in FASN-deficient cells (Fig. 2G). Thus, we show for the first time that FASN contributes to the palmitoylation of endogenous IFITM3. Owing to the observations that IFITM3 is required for an effective IFNβ-mediated anti-influenza response (33McMichael T.M. Zhang L. Chemudupati M. Hach J.C. Kenney A.D. Hang H.C. Yount J.S. The palmitoyltransferase ZDHHC20 enhances interferon-induced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity.J. Biol. Chem. 2017; 292: 21517-21526Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), that palmitoylation of IFITM3 is required for its antiviral activity (33McMichael T.M. Zhang L. Chemudupati M. Hach J.C. Kenney A.D. Hang H.C. Yount J.S. The palmitoyltransferase ZDHHC20 enhances interferon-induced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity.J. Biol. Chem. 2017; 292: 21517-21526Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), and that FASN is required for Alk-4–mediated IFITM3 palmitoylation (Fig. 2G), we sought to determine the effect of FASN expression on IFNβ-mediated inhibition of influenza virus infection. In the absence of IFNβ, FASN expression had no effect on influenza infection (Fig. 2H). However, IFNβ-mediated inhibition of influenza virus infection was significantly decreased in the absence of FASN expression (Fig. 2H), suggesting that FASN-dependent palmitate synthesis likely contributes to the palmitoylation-dependent antiviral activity of IFITM3. Acetyl-CoA is condensed with malonyl-CoA and elongated by FASN to generate palmitate for protein palmitoylation. To generate myristoyl-CoA for myristoylation, palmitate is activated to palmitoyl-CoA by palmitoyl-CoA synthetase, which is then β-oxidized to myristoyl-CoA before it is covalently attached to glycine residues by N-myristoyl transferases (1Resh M.D. Fatty acylation of proteins: The long and the short of it.Prog. Lipid Res. 2016; 63: 120-131Crossref PubMed Scopus (141) Google Scholar, 35Farazi T.A. Waksman G. Gordon J.I. The biology and enzymology of protein N-myristoylation.J. Biol. Chem. 2001; 276: 39501-39504Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). To determine if Alk-4 is metabolized into an FA analog that can selectively label myristoylated proteins, we tested whether a known myristoylated protein, HIV-1 matrix (MA) protein, was labeled upon a 24-h incubation with Alk-4. Human embryonic kidney 293T cells were transfected with Flag-tagged HIV-1 MA or the myristoylation-deficient MA-G2A mutant (MA-G2A) and treated with Alk-4 or Alk-12 (an established chemical reporter of myristoylation). Immunoprecipitation of Flag-tagged MA and subsequent click reaction with azidorhodamine revealed labeling of HIV-1 MA in cells incubated with Alk-4 or Alk-12 (Fig. 3A). The G2A-MA Gag protein, which cannot be myristoylated, was not labeled by Alk-4, indicating myristoylation site–specific labeling of HIV-1 MA protein by Alk-4. Treatment of cells with Fasnall (9Kulkarni M.M. Ratcliff A.N. Bhat M. Alwarawrah Y. Hughes P. Arcos J. Loiselle D. Torrelles J.B. Funderburg N.T. Haystead T.A. Kwiek J.J. Cellular fatty acid synthase is required for late stages of HIV-1 replication.Retrovirology. 2017; 14: 45Crossref PubMed Scopus (21) Google Scholar, 36Alwarawrah Y. Hughes P. Loiselle D. Carlson D.A. Darr D.B. Jordan J.L. Xiong J. Hunter L.M. Dubois L.G. Thompson J.W. Kulkarni M.M. Ratcliff A.N. Kwiek J.J. Haystead T.A. Fasnall, a selective FASN inhibitor, shows potent anti-tumor activity in the MMTV-Neu model of HER2(+) breast cancer.Cell Chem. Biol. 2016; 23: 678-688Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), an FASN inhibitor, abolished Alk-4 labeling of HIV-1 MA protein. As a control, Fasnall treatment did not disrupt HIV-1 MA protein labeling by Alk-12. To confirm the selective labeling of HIV-1 MA protein that we observed with fluorescence-based click reactions, cell lysates were instead reacted with biotin azide. Biotin-conjugated proteins were precipitated with streptavidin agarose, and bound proteins were released with sodium dithionite, which cleaves a diazo linker within the azidobiotin molecule, enabling selective elution of Alk-4 labeled proteins. Eluents were probed for the MA-Flag proteins, and, in the presence of Alk-4, HIV-1 MA protein was recovered. HIV-1 MA protein recovery was diminished both when a myristoylation-deficient HIV-1 MA protein variant was transfected (MA-G2A) (Fig. 3B) and when FASN was inhibited by Fasnall. These results indicate that Alk-4 is metabolized into an FA adduct that is specifically incorporated onto protein myristoylation sites in an FASN-dependent manner, and that can be detected by multiple labeling modalities. To test the utility of Alk-4 as a global indicator of FASN-dependent protein acylation, we incubated the human fibroblast-like cell line HAP1 or an FASN-deficient clone of the HAP1 cells with Alk-4 or a vehicle control (dimethyl sulfoxide [DMSO]). Following metabolic labeling of HAP1 cells with Alk-4, cell lysates were reacted with azidobiotin, and labeled proteins were precipitated as described in Figure 3B. Eluents were then probed for proteins known to be palmitoylated (calnexin) (37Lakkaraju A.K.K. Abrami L. Lemmin T. Blaskovic S. Kunz B. Kihara A. Dal Peraro M. Van Der Goot F.G. Palmitoylated calnexin is a key component of the ribosome-translocon complex.EMBO J. 2012; 31: 1823-1835Crossref PubMed Scopus (106) Google Scholar) or myristoylated (Src) (38Patwardhan P. Resh M.D. Myristoylation and membrane binding regulate c-Src stability and kinase activity.Mol. Cell. Biol. 2010; 30: 4094-4107Crossref PubMed Scopus (117) Google Scholar, 39Resh M.D. Myristylation and palmitylation of Src family members: The fats of the matter.Cell. 1994; 76: 411-413Abstract Full Text PDF PubMed Scopus (585) Google Scholar). In WT HAP1 cells, Alk-4 labeling recovered both calnexin and Src, whereas in FASN-deficient cells, incubation with Alk-4 did not enable calnexin or Src recovery (Fig. 4A). To determine the breadth of proteins recovered from cells incubated with Alk-4, we next used mass spectrometry to identify biotinylated proteins from HAP1 cells with or without Alk-4 and with or without FASN. FASN was only recovered from WT HAP1 cells incubated with Alk-4, consistent with the acyl intermediates formed between FASN and the elongating FA chain (40Wakil S.J. Fatty acid synthase, a proficient multifunctional enzyme.Biochemistry. 1989; 28: 4523-4530Crossref PubMed Scopus (666) Google Scholar). In total, Alk-4 labeling enabled recovery of 264 proteins in an Alk-4- and FASN-dependent manner (Fig. 4B). Of these, 77% (203) have previously been identified in at least one palmitoyl proteome, or they have been experimentally validated to be palmitoylated. These included well-characterized palmitoylation substrates, such as guanine nucleotide-binding protein (G protein), alpha inhibiting 1 (GNA1I) and catenin beta-1 (CTNB1) (Fig. 4C, Tables S1 and S3). Of the remaining proteins that were purified, 17% were predicted to be palmitoylated (e.g., SAM domain and HD domain containing protein 1 [SAMHD1]) and 3% were predicted to be myristoylated (e.g., ribosomal protein S6 kinase alpha [KS6A1]) (41Xie Y. Zheng Y. Li H. Luo X. He Z. Cao S. Shi Y. Zhao Q. Xue Y. Zuo Z. Ren J. GPS-lipid: A robust tool for the prediction of multiple lipid modification sites.Sci. Rep. 2016; 6: 28249Crossref PubMed Scopus (75) Google Scholar, 42Ren J. Wen L. Gao X. Jin C. Xue Y. Yao X. CSS-Palm 2.0: An updated software for palmitoylation sites prediction.Protein Eng. Des. Sel. 2008; 21: 639-644Crossref PubMed Scopus (381) Google Scholar) (Fig. 4C, Tables S1 and S4). This experiment also recovered, in an Alk-4 dependent manner, several enzymes involve" @default.
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