Structure-Function of Protein Deacetylases

描述（由申请人提供）：组蛋白去乙酰化酶（hdac）最初是通过它们能够使组蛋白n端尾部区域内特定赖氨酸残基的epsilon氨基去乙酰化而被发现的，从而促进转录抑制或基因沉默。最近，一些与hdac序列同源的去乙酰化酶被证明可以在体内使非组蛋白靶点去乙酰化，如用于DNA修复调节的p53肿瘤抑制蛋白和用于维持细胞完整性的α -微管蛋白，这表明这些蛋白比转录调节具有更广泛的功能。HDAC蛋白分为三类，采用两种不同的催化机制。I类和II类hdac在催化域中具有相当大的序列同源性，并且不使用辅助因子进行催化。III类hdac属于Sir2蛋白家族，与I/II类hdac具有一级序列和结构差异。此外，Sir2蛋白采用一种新的催化机制，即蛋白质去乙酰化伴随着NAD+水解，产生一种新的o -乙酰基- adp -核糖中间体和烟酰胺。HDAC研究的一个特别令人兴奋的领域涉及它们在人类癌症中的潜在作用，包括人类I类HDAC在急性髓性白血病中的作用，以及III类HDAC在p53肿瘤抑制蛋白的调节中的作用。事实上，HDAC抑制剂目前作为抗癌药物在临床试验中，基于羟肟酸的HDAC抑制剂，如SAHA和TSA，已经在耐受性良好的剂量下显示出对几种不同实体肿瘤的有希望的活性。

DESCRIPTION (provided by applicant): Histone deacetylases (HDACs) were first identified through their ability to deacetylate the epsilon amino group of specific lysine residues within the N-terminal tail regions of histones to promote transcriptional repression or gene silencing. Several deacetylase enzymes that have sequence homology to HDACs have more recently been shown to deacetylate non-histone protein targets in vivo such as the p53 tumor suppressor protein for DNA repair regulation and alpha-tubulin for maintenance of cell integrity, suggesting that these proteins have even broader function than transcriptional regulation. The HDAC proteins fall into three classes and employ two different catalytic mechanisms. Class I and II HDACs show considerable sequence homology within the catalytic domain and do not use a cofactor for catalysis. The class III HDACs belong to the Sir2 protein family and show primary sequence and structural divergence with the class I/II HDACs. In addition, the Sir2 proteins employ a novel catalytic mechanism, whereby protein deacetylation is accompanied by NAD+ hydrolysis generating a novel O-acetyl-ADP-ribose intermediate and nicotinamide. A particularly exciting area of HDAC research relates to their implicated role in human cancer, including the involvement of the human class I HDACs in acute myeloid leukemia and the class III HDACs in the regulation of the p53 tumor suppressor protein. Indeed, HDAC inhibitors are currently in clinical trails as anticancer agents and hydroxamic acid-based HDAC inhibitors, such as SAHA and TSA, have already shown promising activity against several different solid tumors at well-tolerated doses. Despite the important biological role of HDAC proteins and their involvement in human cancer, their mechanism for catalysis, mode of substrate-specific binding, and the biochemical consequence of HDAC deacetylation is poorly understood. This lack of mechanistic information stems from a paucity of structural information on these enzymes. The overall goal of this project is to elucidate the mechanism of HDAC function through a combined structure/function approach on a subset of biologically well-characterized HDAC model proteins. The Specific Aims of the proposal are to (1) Characterize the structure/function of the yeast Sir2 homologue, Hst2; (2) Characterize the structure/function of the bacterial Sir2 homologue, CobB; (3) Determine the crystal structure of archaeal Af1-Sir2 bound to its cognate archaeal chromatin protein substrate, Alba; (4) Determine the structure of the archaeal Af1-Sir2 substrate, Alba; (5) Determine the structure of the class I/II HDACs, human HDAC6 and yeast Hos3. Together, these studies will provide new molecular insights into the mode of catalysis and substrate-specific binding by HDACs, as well as the biochemical consequence of HDAC deacetylation. Moreover, these studies will provide a scaffold for the design of small molecule inhibitors for specific histone deacetylase enzymes that may have applications for the treatment of HDAC-mediated cancers.