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.
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.
Project description:The advances in chemical proteomics have significantly expanded our understanding of the diversity and abundance of fatty-acylated proteins in eukaryotes, and reveal novel functions for these lipid protein modifications. Nonetheless, quantitative comparative proteomic analysis of fatty-acylated proteins in different cellular states is still challenging. To address these limitations, we systematically evaluated different proteomic methods (alk-16 chemical reporter and acyl-RAC) and established robust conditions to selectively and quantitatively profile fatty-acylated proteins in mammalian cells. Using a combination of metabolic labeling with fatty acid chemical reporters, selective chemical enrichment and label-free proteomics, we performed a quantitative analysis of fatty-acylated proteins in naïve and activated macrophages. These studies revealed novel fatty-acylated proteins associated with host immunity that are differently expressed and lipid-modified in different cellular states.
Project description:Given our laboratory interest in IFITM3 S-fatty-acylation and antiviral activity, we sought to directly characterize fatty acids that are covalently attached to the Cys residues of IFITM3. IFITM3 comprises two S-fatty-acylation sites (C71 and C72) in proximity of a intramembrane domain, and another site adjacent to a transmembrane domain (C105). We directly identified the S-fatty-acylation sites of IFITM3, and further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid.
Project description:Given our laboratory interest in IFITM3 S-fatty-acylation and antiviral activity, we sought to directly characterize fatty acids that are covalently attached to the Cys residues of IFITM3. IFITM3 comprises two S-fatty-acylation sites (C71 and C72) in proximity of a intramembrane domain, and another site adjacent to a transmembrane domain (C105). We directly identified the S-fatty-acylation sites of IFITM3 and further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid.
Project description:TEAD transcription factors are responsible for the transcriptional output of Hippo signalling1,2. TEAD activity is primarily regulated by phosphorylation of its coactivators YAP and TAZ3,4. In addition, cysteine palmitoylation has recently been shown to regulate TEAD activity5,6. Here, we report lysine long-chain fatty acylation as a novel posttranslational modification of TEADs. Lysine fatty acylation occurs spontaneously via intramolecular transfer of acyl groups from the proximal acylated cysteine residue. Lysine fatty acylation, like cysteine palmitoylation, contributes to the transcriptional activity of TEADs by enhancing the interaction with YAP and TAZ, but it is more stable than cysteine acylation, suggesting that the lysine fatty-acylated TEAD acts as a “stable active form”. Significantly, lysine fatty acylation of TEAD increased upon Hippo signalling activation, despite a decrease in cysteine acylation. Our results provide new insight into the role of fatty acyl modifications in the regulation of TEAD activity.
Project description:Early-life malnutrition increases adult disease risk in humans, but the causal changes in gene regulation, signaling, and metabolism are unclear. In the roundworm Caenorhabditis elegans, early-life starvation causes well-fed larvae to develop germline tumors and other gonad abnormalities as adults. Furthermore, reduced insulin/IGF signaling (IIS) during larval development suppresses these starvation-induced abnormalities. How early-life starvation and IIS affect adult pathology is unknown. We show that early-life starvation has pervasive effects on adult gene expression that are largely reversed by reduced IIS following recovery from starvation. Early-life starvation increases adult fatty-acid synthetase fasn-1 expression in daf-2 IIS receptor-dependent fashion, and fasn-1/FASN promotes starvation-induced abnormalities. Lipidomic analysis reveals increased levels of phosphatidylcholine in adults subjected to early-life starvation, and supplementation with unsaturated phosphatidylcholine during development suppresses starvation-induced abnormalities. Genetic analysis of fatty-acid desaturases reveals positive and negative effects of desaturation on development of starvation-induced abnormalities. In particular, the delta 3 fatty-acid desaturase fat-1 and the delta 5 fatty-acid desaturase fat-4 inhibit and promote development of abnormalities, respectively. fat-4 is epistatic to fat-1, suggesting that arachidonic acid, or lipids that contain it, promotes development of starvation-induced abnormalities. This work shows that early-life starvation and IIS converge on regulation of adult lipid metabolism, affecting stem-cell proliferation, tumor formation, and additional adult pathologies.
Project description:Early protocells are likely to have arisen from the self-assembly of RNA, peptide, and lipid molecules that were generated and concentrated within geologically favorable environments on the early Earth. The reactivity of these components in a prebiotic environment that supplied sources of chemical energy could have produced additional species with properties favorable to the emergence of protocells. The geochemically plausible activation of amino acids by carbonyl sulfide has been shown to generate short peptides via the formation of cyclic amino acid N-carboxyanhydrides (NCAs). Here, we show that the polymerization of valine-NCA in the presence of fatty acids yields acylated amino acids and peptides via a mixed anhydride intermediate. Notably, Nα-oleoylarginine, a product of the reaction between arginine and oleic acid in the presence of valine-NCA, partitions spontaneously into vesicle membranes and mediates the association of RNA with the vesicles. Our results suggest a potential mechanism by which activated amino acids could diversify the chemical functionality of fatty acid membranes and colocalize RNA with vesicles during the formation of early protocells.
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.
Project description: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.