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Access Type
WSU Access
Date of Award
January 2024
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Mary Kay Pflum
Abstract
Gene expression is regulated by nucleosome remodeling proteins and histone modifications, such as acetylation, phosphorylation, and methylation. Histone acetylation is a dynamic process that involves histone acetyltransferase and histone deacetylases. Histone deacetylases (HDAC) catalyze the removal of acetyl group from the Ɛ-N of acetylated lysine residues in histone tails to form a tight chromatin structure to suppress gene expression. HDAC proteins are linked with many biological functions, cause various diseases, and are therapeutic targets, especially cancers. HDAC6 is related to breast cancer and neurodegenerative diseases specifically. HDAC6 is unique among other HDAC family members due to having tandem catalytic domains. Prior HDAC-related studies have identified unanticipated substrates of HDAC6 catalytic domain 2 (CD2). However, the identification of substrate preference of each catalytic domain has been unsuccessful due to the scarcity of domain-selective inhibitors. The discovery of substrate preference of each catalytic domain will help to uncover the full HDAC6 biological profile in the cell.Here, we applied substrate trapping using HDAC6 mutant C226A to discover catalytic domain 1 (CD1) substrates. We have identified many ubiquitination-related proteins as putative substrates. E3 ligase UBR5 was further validated and revealed that UBR5 trapping is required CD1 mutant and active CD2. Moreover, we documented reduced basal UBR5 expression with elevated HDAC6 overexpression, suggesting a potential protein degradation. Unsuccessful substrate validation of ATP citrate synthase (ACLY) led to the identification of an associated protein of ACLY in the hit list, HUWE1. Given that E3 ligase HUWE1 was further validated as an ACLY-associated protein, the HDAC6 CD1-mediated deacetylation of HUWE1 was discovered. In a second project, prior research documented that HDAC1 associate protein complexes dictate HDAC1 recruitment to nearby histones to regulate gene expression. However, for non-histone substrates, the recruitment mechanism is poorly investigated. Reanalysis of HDAC1 trapping data revealed that most NuRD complex members co-immnuprecipitated as HDAC1-associated proteins. Follow-up co-immunoprecipitation experiment demonstrated that NuRD complex members GATAD2B and MTA2 are isolated with HDAC1 substrates, such as LSD1, RelA, and MSH6. Here, we documented that HDAC1-mediated deacetylation of MSH6 relies on the presence of GATAD2B, which suggests associate protein complexes govern HDAC1 substrate selectivity. In summary, this thesis established the value of trapping mutants to discover the substrate preference of HDAC6 CD1 and selective deacetylation of HDAC1 non-histone substrates. These studies augment our understanding of the unveiled biology of HDAC6 and HDAC1-associated complexes.
Recommended Citation
Ariyaratne, Udana Vijayageetha, "Trapping Mutants To Discover The Substrate Preference Of Hdac Enzymes" (2024). Wayne State University Dissertations. 4032.
https://digitalcommons.wayne.edu/oa_dissertations/4032
