Investigating the Substrate Selectivity and Regulation of Histone Deacetylases

Lysine acetylation regulates thousands of proteins and nearly every cellular process from cell replication to cell death. Dysregulated acetylation has been implicated in diseases including cancer, neurodegenerative disorders, and infectious diseases. For this reason, the enzymes that regulate acetylation, including the histone deacetylases (HDACs), are targeted for drug development, and understanding their biological function is of the utmost importance. Unfortunately, few HDAC-specific substrates have been identified, and how HDACs recognize and select for their substrates, a key aspect of their biological function, is poorly understood. HDAC8, a unique member of class I, is phosphorylated at S39, which affects HDAC8 substrate selectivity in vitro and may be used by the cell to regulate HDAC8 biological function. Measuring HDAC8 phosphomimetic mutant S39E-catalyzed deacetylation of various peptides demonstrates altered HDAC8 activity and importantly substrate selectivity. Structural analyses indicate this alteration is due to changes in the substrate binding pocket and active-site architecture. Moreover, wild-type HDAC8 substrate selectivity is influenced by both substrate sequence and structure in vitro. Comparing HDAC8-catalyzed deacetylation of histone H3 K9ac, K14ac, and K56ac peptides and proteins reveals that protein structure enhances activity from 40- to over 300-fold, and local sequence determines substrate selectivity, particularly in less structured regions. These data support the use of peptide substrates to determine relative activity and to identify HDAC substrates. To expand on these results, HDAC6-catalyzed deacetylation of a library of peptides was tested to develop a structure-based model of HDAC6 activity. The results reveal HDAC6 distinguishes between sequences, catalyzing deacetylation of peptides with kcat/KM values from 10 to 106 M-1s-1. These data demonstrate the usefulness of a prediction model based on peptides. Together, these investigations reveal that phosphorylation, local sequence, and protein structure affect HDAC substrate selectivity and activity in vitro and likely play key roles in the biological function and dysfunction of HDACs in the cell. Finally, development of structure-based models combined with peptide-based experiments can be used to identify HDAC substrate candidates for study in vivo.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/153418/1/krleng_1.pd

Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant

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Recent Advances in Epigenetic Biomarkers and Epigenetic Targeting in Prostate Cancer.

CONTEXT: In addition to genetic alterations, epigenetic alterations play a crucial role during prostate cancer progression. A better understanding of the epigenetic factors that promote prostate cancer progression may lead to the design of rational therapeutic strategies to target prostate cancer more effectively. OBJECTIVE: To systematically review recent literature on the role of epigenetic factors in prostate cancer and highlight key preclinical and translational data with epigenetic therapies. EVIDENCE ACQUISITION: We performed a systemic literature search in PubMed. At the request of the editors, we limited our search to articles published between January 2015 and August 2020 in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Clinical trials targeting epigenetic factors were retrieved from clinicaltrials.gov. EVIDENCE SYNTHESIS: We retrieved 1451 articles, and 62 were finally selected for review. Twelve additional foundational studies outside this time frame were also included. Findings from both preclinical and clinical studies were reviewed and summarized. We also discuss 12 ongoing clinical studies with epigenetic targeted therapies. CONCLUSIONS: Epigenetic mechanisms impact prostate cancer progression. Understanding the role of specific epigenetic factors is critical to determine how we may improve prostate cancer treatment and modulate resistance to standard therapies. Recent preclinical studies and ongoing or completed clinical studies with epigenetic therapies provide a useful roadmap for how to best deploy epigenetic therapies clinically to target prostate cancer. PATIENT SUMMARY: Epigenetics is a process by which gene expression is regulated without changes in the DNA sequence itself. Oftentimes, epigenetic changes influence cellular behavior and contribute to cancer development or progression. Understanding how epigenetic changes occur in prostate cancer is the first step toward therapeutic targeting in patients. Importantly, laboratory-based studies and recently completed and ongoing clinical trials suggest that drugs targeting epigenetic factors are promising. More work is necessary to determine whether this class of drugs will add to our existing treatment arsenal in prostate cancer