Profiling Nonhistone Substrates of the NAD-Dependent Protein Deacetylase SIRT2 Using Site-Specific Azide-Acetyllysine Photochemistry

A, Rashik (2025) Profiling Nonhistone Substrates of the NAD-Dependent Protein Deacetylase SIRT2 Using Site-Specific Azide-Acetyllysine Photochemistry. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Rashik A (20MS133)) 20MS133_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Histone Deacetylases (HDACs), are class of enzymes that remove acetyl groups from lysine residues in histone proteins, influencing chromatin structure and gene expression. Sirtuins (SIRTs), a class III HDACs, are NAD+-dependent protein deacetylases that regulate diverse biological processes, including gene expression, DNA repair, and metabolism. While their role in histone deacetylation is known, their non-histone substrates remain largely unexplored. A comprehensive identification of these non-substrates has remained challenging due to limitations in existing biochemical approaches. This study employed a photo-crosslinking-based interactome profiling (PBIP), a chemo-proteomic strategy to identify transient and dynamic acetylated non-histone proteins. Using amber suppressor mutagenesis, a photo-crosslinkable unnatural amino acids, 4-azido-L-phenylalanine (AzF), was successfully incorporated into the active site of SIRT2. Further the binding affinity of engineered SIRT2 was assessed with its acetylated histone H4 and H3 interacting partners using deacetylation assay. The SIRT2-AzF mutant showed significant crosslinking efficiency, and its ability to capture the cellular proteome. Further proteomic analysis can reveal the novel non-histone substrates of SIRT2 and their implications in various biological pathways. Thus, this work provides a powerful framework for studying SIRT2-mediated protein regulation and opens new avenues for understanding the mechanistic basis of its function in cancer, metabolism, and other pathophysiological conditions. Furthermore, the methodologies developed in this study can serve as a foundation for expanding this approach to engineer and identify the novel substrates of other Sirtuin members, thereby broadening our understanding of their biological and therapeutic significance.

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