Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation

Abstract

Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferoninduced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.fatty-acylation | palmitoylation | PEGylation | influenza virus | IFITM3 P rotein S-fatty acylation describes the covalent attachment of long-chain fatty acids to cysteine (Cys) residues through a thioester bond, which alters the hydrophobicity of diverse proteins and regulates their stability, trafficking, and activity in eukaryotic cells ( Fig. 1A) (1, 2). Cys residues are predominately acylated with palmitic acid (S-palmitoylation), but can also be modified with longer chain and unsaturated fatty acids, thus more generally described as S-fatty acylation (1, 3, 4). The fatty-acylation of Cys residues is regulated by the DHHC-family of protein acyltransferases (5, 6) and different classes of thioesterases (7,8) that are associated with a variety of important physiological pathways and diseases (1). Determining the precise levels of protein S-fatty acylation is therefore crucial for understanding how this dynamic lipid modification is regulated and quantitatively controls specific cellular pathways.Recent methods to detect and enrich S-fatty acylated proteins have facilitated the characterization of key regulatory mechanisms and discovery of new S-fatty acylated proteins (1, 2). For example, alkyne-modified fatty acid chemical reporters have enabled the fluorescent detection and enrichment of metabolically labeled proteins using bioorthogonal ligation methods (Fig. S1A) (9, 10). Alternatively, exploitation of thioester sensitivity to hydroxylamine (NH 2 OH) has enabled selective capture and analysis of S-acylated proteins by acyl-biotin exchange (ABE) (Fig. S1B) (11, 12) or acyl-resin capture (acyl-RAC) (Fig. S1C) (13). However, all of these methods do not readily reveal the fraction of unmodified...

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites

“…Notably, peptides bearing palmitate on C72 were detected with a higher intensity in MS1 and higher confidence/score than peptides bearing palmitate on C71 (Figure 5C, SI Figure S25). These peptides are expected to have similar properties and should have similar solubility and fragmentation behaviors, which suggests that S-palmitoylation of IFITM3 at C72 is more prevalent that S-palmitoylation at C71, consistent with our previous indirect studies using alk-16 and APE 30 . Peptide ASTAKC(+palm)LNIW containing C105 was also identified in the sample with lower confidence (SI Figure 26 and SI Table 8).…”

“…10,48 Our recent analysis of IFITM3 S-fatty-acylation by acyl-PEG exchange (APE) and alk-16 labeling revealed that C72A was the major site of S-fatty-acylation. 30 Since trypsin digest of IFITM3 results in large hydrophobic peptides (>29 amino acids), it appeared advantageous to digest IFITM3 with chymotrypsin, yielding 8–10 amino acid long peptides (SI Figure 18). Due to the predicted hydrophobicity of the lipidated peptides, we first performed preliminary experiments to test whether synthetic S-palmitoylated peptides could be detected by LC-MS/MS.…”

Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress

“…To measure endogenous Hras S-fatty-acylation levels during oxidative stress, we utilized an S-acyl-PEGylation exchange (APE) protocol developed by our laboratory (Supplementary Fig. 13a) 35 . The APE protocol is based on the acyl-biotin exchange protocol 36 , which caps free Cys residues and employs NH 2 OH to cleave thioesters for subsequent modification of thiols with PEG-maleimide reagents to induce a mass shift that is readily detectable by western blot (Supplementary Fig.…”

Human Genetic Determinants of Viral Diseases

“…Recent work in vitro suggests that although this alteration of IFITM3 decreases antiviral activity against influenza virus, its activity against retroviruses is increased (29), thus revealing a potential selective advantage for the SNP that may explain its high prevalence in certain human populations (177). Additionally, given that IFITM3 antiviral activity is highly regulated by at least four posttranslational modifications, polymorphisms in factors that install or remove these modifications and impact influenza virus susceptibility may be uncovered in the future (22–24, 117, 172). …”