A bioorthogonal chemical reporter for fatty acid synthase–dependent protein acylation

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 associated with Smith and Biol. PubMed Scopus Google Scholar), and an to selectively acylated proteins. the bioorthogonal labeling and of protein fatty acylation using click chemistry–compatible of palmitate and have and for of protein X. of click in protein on and Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. M.A. A. Wu P. and of fatty acylated proteins using acids and click Lipid Res. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). The labeling of cells with alkynyl of FAs that can be with to such as or including C. C. D. A. M. D. J. A. A. L. fatty acid by click Biol. PubMed Scopus Google Scholar, M.E. metabolic with acyl-CoA protein 2016; Scopus Google Scholar). Although palmitate and the acylation of proteins by the chemical the role of FASN-dependent de novo–synthesized FAs in cancer, metabolic and viral we that a bioorthogonal reporter of FASN-dependent protein acylation a of the contributions of FASN-dependent protein fatty acylation to protein protein and FASN-mediated Here, we the of alkynyl acetic acid or 5-hexynoic acid a cell-permeable and click that labels proteins acylated by of FASN-mediated de novo FA such as alkynyl palmitate (alkynyl palmitic acid or acid and alkynyl myristic acid or acid are of and activity that are used to identify and myristoylated proteins Zhang M.M. J. E. H.C. of protein with chemical PubMed Scopus Google Scholar, B. Yang H.C. antiviral activity of Biol. 2010; PubMed Scopus Google Scholar, J.C. Chen J. H.C. fatty Biol. 12: PubMed Scopus Google Scholar). and are FA that the of FASN activity or the β-oxidation of palmitate and be used to de novo–synthesized FAs can be protein acylation that a cell-permeable and bioorthogonal of a FASN Alk-4 H.C. chemical for protein 2010; PubMed Scopus Google Scholar), be used to the contributions of FASN-mediated de novo FA synthesis to protein acylation and whether Alk-4 selectively labels we whether a known protein, interferon-induced transmembrane protein 3 labeled a of cells with alkynyl acetate of acid and in with the palmitoylation reporter and labeled as by click of with and Alk-4 also labeled labeling we whether Alk-4 labeling of on its known cysteines B. Yang H.C. antiviral activity of Biol. 2010; PubMed Scopus Google Scholar). to of labeled by or Alk-4 is known to the J. identification of PubMed Scopus (33) Google Scholar), and labeling of by Alk-4 the of Alk-4 to we whether the which has also labeled by to labeled by a in which its cysteines to labeled indicate that Alk-4 is metabolized into a click FA that is protein palmitoylation of Alk-4 at known protein palmitoylation from by click revealed labeling by and Alk-4 and labeling by B, a to labeled by that Alk-4 labeling of on known C, Alk-4 labeling of is by which palmitate from proteins. Alk-4 labeled a its cysteines to that Alk-4 labels on known cysteines kDa molecular standard Alk-4 labeling of a that Alk-4 is metabolized into a long-chain fatty acid by to the of FASN for labeling of in cells with and FASN cells labeled with to FASN in and cells and expression of on Alk-4 labeling of observed in cells and in FASN cells, that FASN contributes to palmitoylation of effective at virus in FASN cells that FASN is required for of an effective response against virus, through for fatty for of fatty acid interferon-induced transmembrane protein proteins are at A. Linder M.E. palmitoylation by a of protein Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar) by to on can with as short as and as long as J. M. J. Smith of fatty acid in the of revealed by click S. A. 2017; PubMed Scopus Google Scholar, Linder M.E. protein but acyl-CoA Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). with than have been on that FAs as L. Van M. The of fatty acids covalently to proteins by can be by exogenously fatty PubMed Google Scholar, H. L. M. E. Brass D.R. of to protein of human Biol. Full Text PDF PubMed Google Scholar, M. H. fatty acid for acylation of viral J. PubMed Scopus Google Scholar, E. J.P. H. H.C. and of protein Proteome Res. PubMed Scopus (26) Google Scholar). the of the for long-chain we to whether labeling of by Alk-4 by which to be among the that can palmitoylation Zhang L. M. J.C. Kenney H.C. The interferon-induced transmembrane protein 3 palmitoylation and antiviral Biol. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). In cells with of a labeling indicate that Alk-4 is metabolized into a long-chain FA that can be used as a by for protein have that palmitoylation is required for its antiviral activity against virus B. Yang H.C. antiviral activity of Biol. 2010; PubMed Scopus Google Scholar, A. S. E. X. H.C. labeling and of protein S. A. 2016; PubMed Scopus Google Scholar). FASN-mediated de novo FA biosynthesis contributes to an antiviral we labeling by Alk-4 in and FASN induced and upregulation independent of FASN expression In cells, Alk-4 in labeling that in cells Thus, we for the that FASN contributes to the palmitoylation of to the that is required for an effective response Zhang L. M. J.C. Kenney H.C. The interferon-induced transmembrane protein 3 palmitoylation and antiviral Biol. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar), that palmitoylation of is required for its antiviral activity Zhang L. M. J.C. Kenney H.C. The interferon-induced transmembrane protein 3 palmitoylation and antiviral Biol. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar), and that FASN is required for palmitoylation we to the of FASN expression on of virus In the of FASN expression on of virus in the of FASN expression that FASN-dependent palmitate synthesis contributes to the antiviral activity of is with and elongated by FASN to palmitate for protein for palmitate is to by which is then to it is covalently attached to 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, T.A. The and of protein Biol. Full Text Full Text PDF PubMed Scopus (420) Google Scholar). Alk-4 is metabolized into an FA analog that can selectively myristoylated we whether a known myristoylated protein, HIV-1 matrix protein, labeled a with cells with HIV-1 or the and with Alk-4 or chemical reporter of of and click with revealed labeling of HIV-1 in cells with Alk-4 or The protein, which be labeled by myristoylation labeling of HIV-1 protein by of cells with (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, Y. Hughes P. Loiselle D. J. M.M. Ratcliff A.N. Kwiek J.J. Haystead T.A. a FASN activity in the of breast Biol. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar), an FASN Alk-4 labeling of HIV-1 a HIV-1 protein labeling by the labeling of HIV-1 protein that we observed with click with proteins with and proteins released with which a the of Alk-4 labeled proteins. for the in the of HIV-1 protein HIV-1 protein both a HIV-1 protein and FASN by indicate that Alk-4 is metabolized into an FA that is protein myristoylation in an FASN-dependent manner, and that can be by labeling the of Alk-4 as a of FASN-dependent protein we the human or an of the cells with Alk-4 or a metabolic labeling of cells with with and labeled proteins as in then for proteins known to be L. S. B. A. M. Van is a of the J. PubMed Scopus Google Scholar) or myristoylated P. M.D. and membrane regulate and Biol. 2010; PubMed Scopus Google Scholar, M.D. and of The of the Full Text PDF PubMed Scopus Google Scholar). In cells, Alk-4 labeling both and in cells, with Alk-4 or the of proteins from cells with we used mass spectrometry to identify proteins from cells with or Alk-4 and with or FASN from cells with with the FASN and the FA S.J. Fatty acid synthase, a PubMed Scopus Google Scholar). In Alk-4 labeling enabled of proteins in an and FASN-dependent have been in at palmitoyl or have been to be palmitoylation such as protein and and the proteins that to be domain and domain protein and to be myristoylated protein Y. Y. Li H. X. Z. S. Y. Zhao Y. Z. Ren J. tool for the prediction of lipid 2016; PubMed Scopus Google Scholar, J. L. X. C. Y. Yao X. for palmitoylation 2008; PubMed Scopus Google Scholar) and This also in an Alk-4 manner, in the of acetate to palmitate and including acetyl-CoA acetyl-CoA and in FA and describe a click chemistry–compatible FASN Alk-4 which selectively labels both and myristoylated HIV-1 proteins. chemistry–compatible Alk-4 of 3H such as long and with low Smith and Biol. PubMed Scopus Google Scholar). While or click can be with that are with the of including mass and Zhang M.M. J. E. H.C. of protein with chemical PubMed Scopus Google Scholar). identification of FASN-dependent protein Alk-4 has more than a FASN because it can be with to purification of FASN-dependent acylated proteins. are the to the of which has been as a chemical tool to protein at it that of the proteins by chemical acylation H.C. chemical for protein 2010; PubMed Scopus Google Scholar). that FASN activity is required for Alk-4 labeling of such as HIV-1 and the of Alk-4 as a reporter of FASN-dependent protein acylation. FASN activity is required for replication of enveloped viruses, including chikungunya (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), 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), J. A. J. metabolic fatty acid synthesis as a target for antiviral 2008; PubMed Scopus Google Scholar), severe acute respiratory syndrome coronavirus M. M. E. N. C. Fatty acid synthase palmitoylation of protein and of with hepatitis Scopus Google Scholar), and many (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, 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, 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). FASN including (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, Y. Hughes P. Loiselle D. J. M.M. Ratcliff A.N. Kwiek J.J. 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Thus, de novo FA biosynthesis is a metabolic during and virus and Alk-4 and its to through the de novo FASN a tool to the role of FASN-dependent protein acylation during FASN-dependent including 5-hexynoic acid from or and synthesized by the to Zhang M.M. J. E. H.C. of protein with chemical PubMed Scopus Google Scholar). in cells obtained from the and in with and and FASN cells obtained from and in with and HIV-1 and by Y. S. Y. C. L. to regulate and 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). and B. Yang H.C. antiviral activity of Biol. 2010; PubMed Scopus Google Scholar) as as J.C. on conserved and cysteines of its and virus Virol. 2013; PubMed Scopus Google Scholar) have been using or to synthesized as Y. Hughes P. Loiselle D. J. M.M. Ratcliff A.N. Kwiek J.J. Haystead T.A. a FASN activity in the of breast Biol. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). virus in for at as T.A. role for in PubMed Scopus Google Scholar, B. Li W. the fate of cells during virus 2011; PubMed Scopus Google Scholar). and FASN cells with or as Zhang L. M. J.C. Kenney H.C. The interferon-induced transmembrane protein 3 palmitoylation and antiviral Biol. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar) and with for of with virus antibodies to of by for with or in with and then and in for on in with complete using the acid used of with and with for at using with and to released by and and the click by of of click of or in of of and of in for at and proteins from the by at for in and proteins on to labeled the in acetic acid, and using on an with and or a with the the using of and for and of biotin-based click cells with with with of in with and for with of click of of of of and of using to and the in and of to of protein in and 200 with with for at in to and in proteins selectively by with using with and on to using standard techniques PubMed Scopus Google Scholar). membrane with in and antibodies from and used at a final of and antibodies from and used at a final of used at a final of and used at a final of using a mass with an in on an 3 using a from The at and the at used for mass using by and the against from independent on Proteome and on with a and a of of and a of in both fatty acylation in proteins in the protein using 3 in at or to be and also against using as in the The mass spectrometry have been to the via the Y. 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