Functional and chemical proteomics analysis to explore lysine posttranslational modifications

Abstract

Metabolomic sensing through protein modification is an important mechanism to regulate protein function, activity, and gene expression. Due to an amine functional group, the lysine residue on proteins harbors a unique reactivity and is sensitive to cell metabolism, environmental changes, and enzyme regulation. However, due to the limitations of the tools and the understanding of the regulation mechanism, the knowledge of lysine modification is still limited. Therefore, with our developed tools, we characterize the three lysine modifications in this study, including lysine sorbylation, 5-hydroxylysine modification, and allysine modification. First, lysine sorbylation is a new type of acylation induced by sorbate, a widely used food preservative. We validated the induction of lysine sorbylation and developed a pan anti-sorbylation antibody to profile the regulation of HDAC. With this antibody, we identified a noncanonical function of HDAC1-3 to induce lysine sorbylation. In addition, we correlated the inflammation response with the HDAC, Nfkb, and sorbylation regulated pathways. This study reveals a novel mechanism for regulating a new lysine modification and inflammation response. Second, protein hydroxylysine has multiple hydroxylation constitutional isomers, including 5-R-hydroxylysine, 5-S-hydroxylysine, 4-hydroxylysine, and 3-hydroxylysine modification. Among the hydroxylysine modifications, 5-S-hydroxylysine is a vital protein modification regulated by JMJD6. However, due to the lack of a globally and constitutionally specific tool for protein modification profiling, the understanding of 5-hydroxylysine modification induced by JMJD6 is restricted. This study developed a periodate oxidation strategy to specifically oxidize the protein 5-hydroxylysine modification. Proteins were further pulled down by hydrazide beads and subjected to trypsin digestion and methoxyamine cleavage for LCMS analysis. With this method, we can discover the regulation of JMJD6 to 5-hydroxylysine modification in a complex system. Third, protein allysine modification is a lysine modification with an aldehyde functional group on the side chain. Due to the uniqueness of the functional group and the low natural abundance, identifying allysine modification is challenging. To overcome this issue, we developed a hydrazide beads capture and D3-methoxyamine release strategy to understand the regulation of allysine modification. This strategy can broaden the understanding of the modification's potential regulation pathway. Collectively, these studies provide insight into the lysine acylation, 5-hydroxylysine modification, and allysine modification regulation pathway.