{"doi":"10.1016/j.jbc.2021.101272","title":"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 ","journal":"Journal of Biological Chemistry","year":2021,"id":183883,"datarank":0.38474240361923057,"base_score":2.5649493574615367,"endowment":2.5649493574615367,"self_citation_contribution":0.38474240361923057,"citation_network_contribution":0.0,"self_endowment_contribution":0.38474240361923057,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":12,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9583,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":69922,"name":"Lizhi Zhang","orcid":"0009-0009-3547-754X","position":1,"is_corresponding":false},{"id":290794,"name":"David R. Loiselle","orcid":"0000-0002-7065-8495","position":2,"is_corresponding":false},{"id":290796,"name":"Timothy Haystead","orcid":"0000-0001-7274-1519","position":3,"is_corresponding":false},{"id":738283,"name":"Menakshi Bhat","orcid":"0000-0002-2586-5778","position":4,"is_corresponding":false},{"id":241044,"name":"Jacob S. Yount","orcid":"0000-0002-6128-4575","position":5,"is_corresponding":false},{"id":380906,"name":"Jesse J. Kwiek","orcid":"0000-0003-4277-8315","position":6,"is_corresponding":false},{"id":738282,"name":"Krithika P. Karthigeyan","orcid":"0000-0002-1808-6688","position":0,"is_corresponding":true}],"reference_count":63,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:48:26.340498Z","pmid":"34606827","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}