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A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation.

ABSTRACT: Mammalian 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 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 SARS-CoV-2, among others. 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. Here 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 labels the cellular protein interferon-induced transmembrane protein 3 (IFITM3) 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.

SUBMITTER: Karthigeyan KP

PROVIDER: S-EPMC8551652 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:Mammalian fatty acid synthase (FASN) is a lipogenic enzyme that catalyzes the formation of the long chain saturated fatty acid palmitate from acetyl and malonyl CoA in the presence of NADPH. Mammalian cells acquire fatty acids through dietary sources or through FASN. Although most mammalian 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 (5-Hexynoic acid, or "Alk-4") that functions as a reporter of FASN-dependent protein acylation. Alk-4 metabolic labeling enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Alk-4 also labeled the palmitoylated host protein IFITM3 (Interferon inducible transmembrane protein-3), a restriction factor for Influenza, and the myristoylated HIV-1 MA (Matrix) protein. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.

2022-02-15 | PXD028244 | Pride

Fatty-acylation in RAW264.7 - Bioorthogonal Chemical Reporters for Monitoring Unsaturated Fatty-Acylated Proteins

Project description:Dietary unsaturated fatty acids beneficially affect human health, in part by modulating the immune system, but the mechanism is not completely understood. Given that unsaturated fatty acids have been shown to be covalently incorporated into a small subset of proteins, we designed three alkyne-tagged chemical reporters of unsaturated fatty acids, alk-16:1, alk-17:1 and alk-18:1, to explore the generality and diversity of this protein modification. Following cell lysis, proteins labelled with the reporters could be captured by azido-functionalized reagents via CuAAC for fluorescence detection or enrichment for proteomics analysis. These reporters label many proteins in mammalian cells and can be incorporated site-specifically, notably on Cys residues. Quantitative proteomics analysis (n= 4 biological replicates) of LPS/IFN-gamma stimulated RAW264.7 labelled with oleic acid (control), alk-16 (palmitic acid chemical reporter), alk-16:1, alk-17:1 and alk-18:1, revealed that unsaturated fatty acids modify similar protein targets to saturated fatty acids, including several immune proteins. Interestingly, some proteins can be differentially labeled with unsaturated and saturated fatty acid.

2018-10-26 | PXD003545 | Pride

A Bioorthogonal Chemical Reporter of Viral Infection.

Project description:Pathogen-selective labeling was achieved by using the novel gemcitabine metabolite analogue 2'-deoxy-2',2'-difluoro-5-ethynyluridine (dF-EdU) and click chemistry. Cells infected with Herpes Simplex Virus-1 (HSV-1), but not uninfected cells, exhibit nuclear staining upon the addition of dF-EdU and a fluorescent azide. The incorporation of the dF-EdU into DNA depends on its phosphorylation by a herpes virus thymidine kinase (TK). Crystallographic analyses revealed how dF-EdU is well accommodated in the active site of HSV-1 TK, but steric clashes prevent dF-EdU from binding human TK. These results provide the first example of pathogen-enzyme-dependent incorporation and labeling of bioorthogonal functional groups in human cells.

| S-EPMC7159771 | biostudies-literature

A Bioorthogonal Chemical Reporter of Viral Infection.

| S-EPMC7159598 | biostudies-literature

Bioorthogonal pro-metabolites for profiling short chain fatty acylation.

Project description:Short chain fatty acids (SCFAs) play a central role in health and disease. One function of these signaling molecules is to serve as precursors for short chain fatty acylation, a class of metabolically-derived posttranslational modifications (PTMs) that are established by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). Via this mechanism, short chain fatty acylation serves as an integrated reporter of metabolism as well as KAT and KDAC activity, and has the potential to illuminate the role of these processes in disease. However, few methods to study short chain fatty acylation exist. Here we report a bioorthogonal pro-metabolite strategy for profiling short chain fatty acylation in living cells. Inspired by the dietary component tributyrin, we synthesized a panel of ester-caged bioorthogonal short chain fatty acids. Cellular evaluation of these agents led to the discovery of an azido-ester that is metabolized to its cognate acyl-coenzyme A (CoA) and affords robust protein labeling profiles. We comprehensively characterize the metabolic dependence, toxicity, and histone deacetylase (HDAC) inhibitor sensitivity of these bioorthogonal pro-metabolites, and apply an optimized probe to identify novel candidate protein targets of short chain fatty acids in cells. Our studies showcase the utility of bioorthogonal pro-metabolites for unbiased profiling of cellular protein acylation, and suggest new approaches for studying the signaling functions of SCFAs in differentiation and disease.

| S-EPMC5885804 | biostudies-literature

A bioorthogonal chemical reporter for the detection and identification of protein lactylation.

Project description:l-Lactylation is a recently discovered post-translational modification occurring on histone lysine residues to regulate gene expression. However, the substrate scope of lactylation, especially that in non-histone proteins, remains unknown, largely due to the limitations of current methods for analyzing lactylated proteins. Herein, we report an alkynyl-functionalized bioorthogonal chemical reporter, YnLac, for the detection and identification of protein lactylation in mammalian cells. Our in-gel fluorescence and chemical proteomic analyses show that YnLac is metabolically incorporated into lactylated proteins and directly labels known lactylated lysines of histones. We further apply YnLac to the proteome-wide profiling of lactylation, revealing many novel modification sites in non-histone proteins for the first time. Moreover, we demonstrate that lactylation of a newly identified substrate protein PARP1 regulates its ADP-ribosylation activity. Our study thus provides a powerful chemical tool for characterizing protein lactylation and greatly expands our understanding of substrate proteins and functions of this new modification.

| S-EPMC9132054 | biostudies-literature

Bifunctional fatty acid chemical reporter for analyzing S-palmitoylated membrane protein-protein interactions in mammalian cells.

Project description:Studying the functions of S-palmitoylated proteins in cells can be challenging due to the membrane targeting property and dynamic nature of protein S-palmitoylation. New strategies are therefore needed to specifically capture S-palmitoylated protein complexes in cellular membranes for dissecting their functions in vivo. Here we present a bifunctional fatty acid chemical reporter, x-alk-16, which contains an alkyne and a diazirine, for metabolic labeling of S-palmitoylated proteins and photo-cross-linking of their involved protein complexes in mammalian cells. We demonstrate that x-alk-16 can be metabolically incorporated into known S-palmitoylated proteins such as H-Ras and IFITM3, a potent antiviral protein, and induce covalent cross-linking of IFITM3 oligomerization as well as its specific interactions with other membrane proteins upon in-cell photoactivation. Moreover, integration of x-alk-16-induced photo-cross-linking with label-free quantitative proteomics allows identification of new IFITM3 interacting proteins.

| S-EPMC4303541 | biostudies-literature

Novel fatty acid acylation of lens integral membrane protein aquaporin-0.

Project description:Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.

| S-EPMC3831505 | biostudies-literature

Human fatty acid synthase: assembling recombinant halves of the fatty acid synthase subunit protein reconstitutes enzyme activity.

Project description:Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr approximately 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the beta-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 A of the pantetheinyl-SH of the second subunit (where the malonyl group is bound). This arrangement generates two active centers for fatty acid synthesis and predicts that if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site. We cloned, expressed, and purified catalytically active thioredoxin (TRX) fusion proteins of the NH2-terminal half of the human FAS subunit protein (TRX-hFAS-dI; residues 1-1,297; Mr approximately 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296-2,504; Mr approximately 155). Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH resulted in the synthesis of long-chain fatty acids. The rate of synthesis was dependent upon the presence of both recombinant proteins and reached a constant level when they were present in equivalent amounts, indicating that the reconstitution of an active fatty acid-synthesizing site required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to be the most abundant with small amounts of palmitate and stearate, possibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state. The successful reconstitution of the human FAS activity from its domain I and domains II and III fully supports our model for the structure-function relationship of FAS in animal tissues.

| S-EPMC24928 | biostudies-literature

Bioorthogonal Chemical Reporters for Monitoring Unsaturated Fatty-Acylated Proteins.

Project description:Dietary unsaturated fatty acids, such as oleic acid, have been shown to be covalently incorporated into a small subset of proteins, but the generality and diversity of this protein modification has not been studied. We synthesized unsaturated fatty-acid chemical reporters and determined their protein targets in mammalian cells. The reporters can induce the formation of lipid droplets and be incorporated site-specifically onto known fatty-acylated proteins and label many proteins in mammalian cells. Quantitative proteomics analysis revealed that unsaturated fatty acids modify similar protein targets to saturated fatty acids, including several immunity-associated proteins. This demonstrates that unsaturated fatty acids can directly modify many proteins to exert their unique and often beneficial physiological effects in vivo.

| S-EPMC5050088 | biostudies-literature

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