Structure and Function of the SAGA Deubiquitinating Module

描述（由申请人提供）：真核基因的调控依赖于一系列复杂的事件，包括翻译后组蛋白修饰的添加和去除，这在基因激活中起着核心作用。这些生化事件是由包含组蛋白修饰酶的大型多蛋白复合物以及控制酶活性、识别染色质底物并与转录和RNA加工机制成分相关的其他亚基精心策划的。虽然RNA聚合酶、基础转录因子和dna结合蛋白的结构研究取得了巨大进展，为真核生物转录提供了重要的机制见解，但由于缺乏结构信息，对共激活物复合物在这一过程中如何起作用的理解滞后。该项目的长期目标是在分子细节上揭示共激活物复合体中的不同亚基如何协同工作以修改其正确的染色质靶标并与转录装置的其他组分相互作用。这项拨款申请的中心是SAGA共激活因子复合物，它使组蛋白尾部乙酰化，使组蛋白H2B去泛素化，并在转录起始和延伸中发挥作用。包含SAGA复合物的21种蛋白质从酵母到人类广泛保守，并被组织成不同的子模块。去泛素化（DUB）模块，从组蛋白H2B的Lys123中去除单泛素，包含四个蛋白：Ubp8， Sus1， Sgf11和Sgf73。虽然Ubp8含有一个泛素水解酶结构域，但其他三个DUB亚基必须全部与Ubp8结合才能激活其酶活性。目前尚不清楚这四种蛋白的结合是如何增强去泛素化活性的，也不知道这种酶复合物是如何靶向其组蛋白底物的。最近Ubp8/Sgf11/Sus1/Sgf73与泛素醛结合的高分辨率结构揭示了DUB模块亚基的不寻常缠绕排列，现在为详细研究酶激活和靶向组蛋白奠定了基础。目的1说明Ubp8是如何被其他DUB亚基激活的。生化和x射线晶体学研究的结合将用于测试关于每个亚基对Ubp8中催化残基的正确取向和泛素结合的贡献的特定假设。目的2探讨DUB模块如何通过与H2A/H2B二聚体或核小体DNA的特定相互作用靶向核小体底物。我们将利用新的方法来产生化学泛素化组蛋白，在一个明确的，均匀的系统中进行定量研究。酶动力学测定、结构测定和体内测定将用于确定SAGA DUB模块和核小体中负责适当底物识别的决定因素。从这些研究中出现的发现将与人类SAGA复合体直接相关，其亚基和组成与酵母复合体高度相似。由于人类ubb模块蛋白的缺陷与脊髓小脑性共济失调7型有直接关系，而人类Ubp8同源物Usp22是一种癌症干细胞标志物，我们的研究将为DUB功能障碍如何引起人类疾病提供直接的见解。

DESCRIPTION (provided by applicant): The regulation of eukaryotic genes depends upon a complex series of events that include the addition and removal of post-translational histone modifications, which play a central role in gene activation. These biochemical events are orchestrated by large multiprotein complexes containing histone-modifying enzymes, along with other subunits that control enzyme activity, recognize chromatin substrates, and associate with components of the transcriptional and RNA processing machinery. While great advances in structural studies of RNA polymerase, basal transcription factors and DNA-binding proteins have contributed important mechanistic insights into eukaryotic transcription, an understanding of how coactivator complexes function in this process has lagged due to a lack of structural information. The long-term goal of this project is to uncover in molecular detail how the different subunits in coactivator complexes work in concert to modify their correct chromatin targets and interact with other components of the transcription apparatus. This grant proposal centers on the SAGA coactivator complex, which acetylates histone tails and deubiquitinates histone H2B, and plays a role in both transcription initiation and elongation. The 21 proteins that comprise the SAGA complex are widely conserved from yeast to humans, and are organized into distinct sub-modules. The deubiquitinating (DUB) module, which removes monoubiquitin from Lys123 of histone H2B, contains four proteins: Ubp8, Sus1, Sgf11, and Sgf73. Although Ubp8 contains a ubiquitin hydrolase domain, the other three DUB subunits must all bind to Ubp8 to activate its enzymatic activity. It is not understood how the association of all four proteins potentiates the deubiquitinating activity, nor is it known how this enzyme complex is targeted to its histone substrate. The recent high-resolution structure of Ubp8/Sgf11/Sus1/Sgf73 bound to ubiquitin aldehyde revealed an unusual intertwined arrangement of DUB module subunits and now sets the stage for detailed investigations into enzyme activation and targeting to histones. Aim 1 addresses how Ubp8 is activated by the other DUB subunits. A combination of biochemical and x-ray crystallographic studies will be used to test specific hypotheses regarding the contribution of each subunit to the correct orientation of catalytic residues in Ubp8 and to ubiquitin binding. Aim 2 addresses how the DUB module is targeted to nucleosomal substrates through specific interactions with either H2A/H2B dimers or with nucleosomal DNA. We will take advantage of new methods to generate chemically ubiquitinated histones to carry out quantitative studies in a defined, homogeneous system. Assays of enzyme kinetics, structure determination and in vivo assays will be used to identify the determinants in both the SAGA DUB module and in nucleosomes that are responsible for proper substrate recognition. The findings that emerge from these studies will be of direct relevance to the human SAGA complex, whose subunits and composition is highly similar to the yeast complex. Since defects in a human DUB module protein are directly implicated in Spinocerebellar Ataxia type 7 and the human Ubp8 homologue, Usp22, is a cancer stem cell marker, our studies will provide immediate insights into how DUB dysfunction gives rise to human disease. PUBLIC HEALTH RELEVANCE: Since defects in a human DUB module protein are directly implicated in Spinocerebellar Ataxia type 7 and the human Ubp8 homologue, Usp22, is a cancer stem cell marker, our studies will provide immediate insights into how DUB dysfunction gives rise to human disease. Understanding the mechanisms by which the Ubp8 enzyme is activated will help guide the design of therapeutic agents that can modulate the activity of deubiquitinating enzyme. Since the success of the proteasomal inhibitor, Bortezomib (VelcadeTM), in treating multiple myleloma, deubiquitinating enzymes are now viewed as another promising target for anti-cancer drugs that target ubiquitin-mediated protein degradation.