Structure-Function of Protein Deacetylases

描述(由申请人提供)：组蛋白脱乙酰酶(HDAC)最初是通过对组蛋白N-末端尾部区域中特定赖氨酸残基的epsilon氨基进行脱乙酰化来促进转录抑制或基因沉默而鉴定的。几种与HDACs序列同源的脱乙酰酶最近被证明可以在体内去乙酰化非组蛋白靶标，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，这表明这些蛋白质具有比转录调节更广泛的功能。HDAC蛋白分为三类，采用两种不同的催化机制。I类和II类HDAC在催化区域内显示出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具有初级序列和结构上的差异。此外，Sir2蛋白采用了一种新的催化机制，即蛋白质的脱乙酰化伴随着NAD+的水解生成一种新的O-乙酰-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牵连作用，包括人类I类HDAC参与急性髓系白血病和III类HDAC参与P53肿瘤抑制蛋白的调节。事实上，HDAC抑制剂目前正在进行临床试验，因为抗癌药物和基于异羟肟酸的HDAC抑制剂，如SAHA和TSA，已经在耐受性良好的剂量下对几种不同的实体肿瘤显示出良好的活性。 尽管HDAC蛋白具有重要的生物学作用及其在人类癌症中的作用，但对其催化机制、底物特异性结合方式以及HDAC脱乙酰化的生化后果仍知之甚少。这种机制信息的缺乏源于缺乏关于这些酶的结构信息。该项目的总体目标是通过对具有良好生物学特性的HDAC模型蛋白的子集进行结构/功能组合的方法来阐明HDAC功能的机制。该建议的具体目的是(1)表征酵母Sir2同源物HST2的结构/功能；(2)表征细菌Sir2同源物Cobb的结构/功能；(3)确定与其同源古菌染色质蛋白底物alba结合的古生菌AF1-Sir2的晶体结构；(4)确定古生菌AF1-Sir2底物alba的结构；(5)确定I/II类HDACs、人HDAC6和酵母Hos3的结构。综上所述，这些研究将为HDAC的催化和底物特异性结合模式以及HDAC脱乙酰化的生化后果提供新的分子见解。此外，这些研究将为设计特定组蛋白脱乙酰基酶的小分子抑制剂提供一个支架，这些酶可能应用于HDAC介导的癌症的治疗。

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.