Structure and Function of the SAGA Deubiquitinating Module

描述(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移除，组蛋白修饰在基因激活中发挥核心作用。这些生化事件是由包含组蛋白修饰酶的大型多蛋白复合体以及其他亚基协调的，这些亚基控制酶活性，识别染色质底物，并与转录和RNA处理机制的组件相关联。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很大进展，为真核转录提供了重要的机制见解，但由于缺乏结构信息，对辅助激活物如何在这一过程中发挥作用的理解一直滞后。该项目的长期目标是从分子细节上揭示共激活因子复合体中的不同亚基如何协同工作，修改其正确的染色质靶标，并与转录装置的其他组件相互作用。这项拨款提案集中在SAGA共激活复合体上，它对组蛋白尾部进行乙酰化，并去泛素化组蛋白H2B，并在转录起始和延伸中发挥作用。组成SAGA复合体的21种蛋白质从酵母到人类都被广泛保守，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，包含四种蛋白质：Ubp8、Sus1、Sgf11和Sgf73。虽然Ubp8含有泛素水解酶结构域，但其他三个DUB亚基都必须与Ubp8结合才能激活其酶活性。目前还不清楚所有四种蛋白质的结合如何增强去泛素活性，也不知道这种酶复合体是如何靶向其组蛋白底物的。最近与泛素醛结合的Ubp8/Sgf11/Sus1/Sgf73的高分辨结构揭示了一种不寻常的交织排列的Dub模块亚基，现在为详细研究酶的激活和靶向组蛋白奠定了基础。目标1阐述了Ubp8是如何被其他Dub亚基激活的。将使用生化和X射线结晶学研究相结合的方法来测试关于每个亚基对Ubp8中催化残基的正确方向和泛素结合的贡献的具体假设。目的2阐述DUB模块如何通过与H_2A/H_2B二聚体或与核小体DNA的特定相互作用而靶向于核小体底物。我们将利用新的方法来产生化学泛素化的组蛋白，以在一个明确的、均一的系统中进行定量研究。将使用酶动力学分析、结构测定和体内分析来确定SAGA DUB模块和核小体中负责正确底物识别的决定因素。这些研究的发现将与人类佐贺复合体直接相关，人类佐贺复合体的亚基和组成与酵母复合体高度相似。由于人类DuB模块蛋白的缺陷直接与脊髓小脑性共济失调7型有关，而人类Ubp8同源物Usp22是癌症干细胞标记物，我们的研究将为DuB功能障碍如何导致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.