Structure and Function of the SAGA Deubiquitinating Module

SAGA去泛素化模块的结构和功能

基本信息

批准号：
8327136
负责人：
Cynthia Wolberger
金额：
$ 24.93万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2015-08-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8327136
关键词：
Acetylation Active Sites Address Affect Antineoplastic Agents Applications Grants Binding Binding Sites Biochemical Biological Assay Bortezomib Cells Chromatin Cleaved cell Complex DNA DNA-Binding Proteins DNA-Directed RNA Polymerase Defect Deubiquitinating Enzyme Deubiquitination Drug Delivery Systems Enzyme Activation Enzyme Kinetics Enzymes Event Excision Functional disorder Gene Activation General Transcription Factors Genes Genetic Transcription Goals Histone H2B Histones Homologous Gene Human Human Activities Hydrolase Individual Inherited Investigation Light Mediating Methods Molecular Multienzyme Complexes Multiprotein Complexes Mutagenesis Mutation Neurodegenerative Disorders Nucleosomes Peptides Play Positioning Attribute Process Protease Domain Proteins RNA Processing Recruitment Activity Regulation Resolution Role SAGA Series Side Signal Transduction Solutions Specificity Staging Structure Substrate Interaction System Tail Telomere Maintenance Testing Therapeutic Agents Transcription Elongation Transcription Initiation Type 7 Spinocerebellar Ataxia Ubiquitin Ubiquitination Work Yeasts Zinc Fingers base cancer stem cell design dimer enzyme activity enzyme substrate histone modification human disease in vivo inhibitor/antagonist insight interest man mutant polyglutamine protein degradation success ubiquitin-aldehyde ubiquitin-specific protease

项目摘要

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.

描述（由申请人提供）：真核基因的调节取决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，这些修饰在基因激活中发挥核心作用。这些生化事件是由含有组蛋白修饰酶的大型多蛋白复合物以及控制酶活性、识别染色质底物以及与转录和 RNA 加工机器组件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，为真核转录的机制提供了重要的见解，但由于缺乏结构信息，对共激活复合物如何在此过程中发挥作用的理解仍然滞后。该项目的长期目标是在分子细节上揭示共激活复合物中的不同亚基如何协同工作以修改其正确的染色质目标并与转录装置的其他组件相互作用。该资助提案以 SAGA 共激活因子复合物为中心，该复合物乙酰化组蛋白尾部并使组蛋白 H2B 去泛素化，并在转录起始和延伸中发挥作用。构成 SAGA 复合体的 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) 模块可从组蛋白 H2B 的 Lys123 中去除单泛素，包含四种蛋白质：Ubp8、Sus1、Sgf11 和 Sgf73。尽管 Ubp8 含有泛素水解酶结构域，但其他三个 DUB 亚基必须全部与 Ubp8 结合才能激活其酶活性。目前尚不清楚所有四种蛋白质的结合如何增强去泛素化活性，也不知道这种酶复合物如何靶向其组蛋白底物。最近与泛素醛结合的 Ubp8/Sgf11/Sus1/Sgf73 的高分辨率结构揭示了 DUB 模块亚基的不寻常的交织排列，现在为详细研究酶激活和靶向组蛋白奠定了基础。目标 1 解决 Ubp8 如何被其他 DUB 亚基激活。将结合生化和 X 射线晶体学研究来测试有关每个亚基对 Ubp8 中催化残基的正确方向和泛素结合的贡献的具体假设。目标 2 解决 DUB 模块如何通过与 H2A/H2B 二聚体或核小体 DNA 的特定相互作用来靶向核小体底物。我们将利用新方法生成化学泛素化组蛋白，在明确的均质系统中进行定量研究。酶动力学、结构测定和体内测定将用于鉴定 SAGA DUB 模块和核小体中负责正确底物识别的决定因素。这些研究的结果将与人类 SAGA 复合物直接相关，其亚基和组成与酵母复合物高度相似。由于人类 DUB 模块蛋白的缺陷与 7 型脊髓小脑共济失调直接相关，而人类 Ubp8 同源物 Usp22 是一种癌症干细胞标记物，因此我们的研究将提供有关 DUB 功能障碍如何引起人类疾病的直接见解。