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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
Histone Deacetylase (HDAC) enzymes remove acetyl groups from acetyl-lysine containing substrate proteins to regulate numerous cellular events, such as cell cycle progression, apoptosis, and cell proliferation. The clear role of HDAC proteins in cancer has led the development of HDAC inhibitors as anticancer therapeutics. Among the eleven-metal dependent HDAC proteins, class IIa HDAC isoforms (HDAC4, 5, 7, and 9) are enzymatically inactive in deacetylation reactions, and are pseudodeacetylases, due to mutation of a catalytic Tyr to His. Instead, class IIa HDAC proteins regulate histone deacetylation through a multi-protein complex that includes nuclear receptor corepressor NCoR/SMRT and active HDAC3. This multi-protein complex allows class IIa HDAC proteins to act as scaffolds to recruit active HDAC3 for deacetylation of acetylated lysines. But, only HDAC4, HDAC5 and HDAC7 associate with NCoR-HDAC3, which suggests that the HDAC9 mechanism of function might be different. Even with this known mechanism, the purpose of the inactive catalytic domain of class IIa HDAC isoforms in the scaffolding activity is unknown. Hence, class IIa HDACs are hypothesized to be “reader” proteins that bind to acetylated lysine residues on target proteins to cause a downstream biological function. However, the roles of HDAC4, 5, 7, and 9 as readers are yet to be explored. Here, we focused on studying scaffolding activities of two pseudodeacetylases HDAC7 and HDAC9.Recent evidence from the Pflum lab showed that the inactive pseudo-catalytic site of HDAC7 acts as a “reader” that binds acetyllysine-containing protein clients to control transcription by disrupting scaffolding to active HDAC3-NCoR complex. While prior work focused on AR (androgen receptor), here ER-⍺ (estrogen receptor alpha) transcription factor also showed acetylation-dependent HDAC7-NCoR-HDAC3 binding. Further, physiologically relevant breast cancer cells demonstrated HDAC7-mediated gene expression corroborating the reader function via NCoR-HDAC3 complex. These data substantiate HDAC7 as a reader of acetylation adding further mechanistic insights into the epigenetic roles of HDAC7. In a second project, we focused on HDAC9, which is one of the poorly studied pseudodeacetylases. Based on the reader hypothesis, we aimed to identify active site binders, scaffolding mediators, and associate proteins of HDAC9 using SAHA-dependent binder trapping strategy. The proteomics analysis revealed many potential deacetylase substrates, reader mediators, and associate proteins of HDAC9. Even though validation of candidate SAHA-dependent binders identified from the proteomics analysis was challenging, we discovered a novel interaction between HDAC9 and BAG2 (BAG family molecular chaperone regulator 2) which was not reported previously. Future studies will unveil the biological significance of HDAC9-BAG2 interaction. In summary, this dissertation work provided further evidence to establish HDAC7 reader function, Also, work documented here conveyed potential interactors of HDAC9 which will facilitate future research to understand unanticipated epigenetic and non-epigenetic roles of HDAC9. Finally, these studies broaden the insights into pseudodeacetylase biology and would encourage further investigations to gain more knowledge on class IIa HDAC proteins.
Recommended Citation
Kodikara, Ishadi, "Scaffolding Activities Of Pseudodeacetylases" (2024). Wayne State University Dissertations. 4138.
https://digitalcommons.wayne.edu/oa_dissertations/4138
