Studies on the mechanism of histone deacetylase enzymes

Abstract

The nucleosome core particle, which is the fundamental building block of chromatin, is composed of 146–147 base pairs of DNA wrapped around a histone octomer containing one H3–H4 tetramer and two H2A–H2B dimers (Luger 2003). Histone proteins are the sites of many different types of posttranslational modifications including acetylation, phosphorylation, methylation, ubiquitination, sumolation, and ADP-ribosylation (Khorasanizadeh 2004). Dynamic acetylation of the ϵ-amino group of specific lysine residues on the N-terminal tails of histones is achieved by histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes (Grunstein 1997). Hyperacetylation is associated with euchromatin, which has a more open conformation allowing for increased gene expression. On the other hand, hypoacetylation is found in condensed heterochromatin and is coupled with repression of transcriptional activity (de Ruijter, van Gennip et al. 2003; Hake, Xiao et al. 2004). HDAC enzymes have been organized into three phylogenetic classes based on sequence homology, inhibitor sensitivity, and cofactor necessity (de Ruijter, van Gennip et al. 2003). The class I and II zinc-dependent HDACs participate in cell cycle control and growth regulation, and consequently their inhibitors have demonstrated the ability to arrest tumor cell growth, induce differentiation, and cause apoptosis (Marks, Miller et al. 2003). HDACs play integral roles in cancer gene regulation and cancer proliferation and as a result, HDAC inhibitors represent promising new chemotherapies for the treatment of certain hematological malignancies like acute leukemias, non-Hodgkins lymphoma, and cutaneous T-cell lymphoma, as well as for tumors of the breast, colon, lung, prostate and stomach (Huang, Sloan et al. 2003; Marks, Miller et al. 2003; Hake, Xiao et al. 2004; Somech, Izraeli et al. 2004). Thorough characterization of HDAC activity is paramount not only to our understanding of gene-specific transcriptional regulation, but indispensable to our complete understanding of cancer pathology. Despite the vital role played by class I and II zinc-dependent HDAC enzymes in gene expression and cancer, the exact mechanism of their catalytic activity remains unknown. In this dissertation, a continuous spectrophotomeric assay for monitoring HDAC activity will be described. Details of the kinetic and chemical mechanism of HDLP, a histone deacetylase-like protein from A. aeolicus will be presented. Mutational analysis of catalytically important residues of HDLP will be discussed to provide further insight into the mechanism of this intriguing class of enzymes