Browsing by Subject "Post-translational Modification"

Protein modifications modulate nearly every aspect of cell biology in organisms ranging from Archaea to Eukaryotes. The earliest evidence of covalent protein modifications was found in the early 20th century by studying the amino acid composition of proteins by chemical hydrolysis. These discoveries challenged what defined a canonical amino acid. The advent and rapid adoption of mass spectrometry-based proteomics in the latter part of the 20th century enabled a veritable explosion in the number of known protein modifications, with over 500 discrete modifications counted today. Now, new computational tools in data science, machine learning, and artificial intelligence are poised to allow researchers to make significant progress discovering new protein modifications and determining their function. Lysine acetylation is one of the most well-known post translational modifications. Acetylation is not limited to lysine with acetylation of serine and threonine having also been reported in the literature. Lysine acetylation is known to occur both enzymatically and non-enzymatically. Cysteine is a reactive amino acid central to the catalytic activities of many enzymes. Given the highly reactive nature of the cysteine side-chain, non-enzymatic acetylation of cysteine would be expected to be more favorable than non-enzymatic acetylation of lysine. Cysteine is also a common target of post-translational modifications (PTMs), such as palmitoylation. This long-chain acyl PTM can modify cysteine residues and induce changes in protein sub-cellular localization. Transfer of an acetyl moiety from the side-chain of cysteine to the side-chain of lysine has been shown in vitro. Cysteine side-chain acetylation has never been shown in vivo. We hypothesized that cysteine could also be modified by short-chain acyl groups, such as cysteine S-acetylation. To test this, we developed sample preparation and non-targeted mass spectrometry protocols to analyze the mouse liver proteome for cysteine acetylation. Our findings revealed hundreds of sites of cysteine acetylation across multiple tissue types, revealing a previously uncharacterized cysteine acetylome. The cysteine acetylome shows distinct patterns in different sub-cellular compartments and is most abundant in the cytoplasm. Cysteine acetylation is present in all tissue types tested and has tissue-specific acetylome patterns. Metabolic stress led to targeted changes in the cysteine acetylome of BAT. Acetylation of the active site cysteines of GAPDH led to a sharp reduction in activity. This study uncovers a novel aspect of cysteine biochemistry, highlighting short-chain modifications alongside known long-chain acyl PTMs. These findings enrich our understanding of the landscape of acyl modifications and suggest new research directions in enzyme activity regulation and cellular signaling in metabolism.