Structure and Function of the SAGA Deubiquitinating Module

描述（由申请人提供）：真核基因的调控取决于一系列复杂的事件，包括添加和去除翻译后组蛋白修饰，其在基因活化中起核心作用。这些生化事件是由含有组蛋白修饰酶的大的多蛋白复合物协调的，沿着的还有控制酶活性、识别染色质底物并与转录和RNA加工机制的组分相关的其他亚基。虽然在RNA聚合酶，基础转录因子和DNA结合蛋白的结构研究的巨大进步，真核生物转录的机制的见解作出了重要贡献，了解如何辅激活复合物在这一过程中的功能已经滞后，由于缺乏结构信息。该项目的长期目标是在分子细节上揭示辅激活因子复合物中的不同亚基如何协同工作以修饰其正确的染色质靶点并与转录装置的其他组件相互作用。这项拨款提案集中在佐贺辅激活因子复合物上，该复合物使组蛋白尾部乙酰化并使组蛋白H2 B去泛素化，并在转录起始和延伸中发挥作用。组成佐贺复合物的21种蛋白质从酵母到人类都是广泛保守的，并且被组织成不同的子模块。去泛素化（DUB）模块，从组蛋白H2 B的Lys 123中去除monoubiquitin，包含四种蛋白质：Ubp 8，Sus 1，Sgf 11和Sgf 73。虽然Ubp 8含有泛素水解酶结构域，但其他三个DUB亚基必须全部结合Ubp 8以激活其酶活性。目前还不清楚所有四种蛋白质的缔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。最近的高分辨率结构的Ubp 8/Sgf 11/Sus 1/Sgf 73结合到泛素醛揭示了一个不寻常的交织排列的DUB模块亚基，现在设置的阶段，详细研究酶的激活和靶向组蛋白。目的1解决了Ubp 8如何被其他DUB亚基激活。生物化学和X射线晶体学研究的组合将被用来测试特定的假设，每个亚基的贡献，在Ubp 8和泛素结合的催化残基的正确方向。目的2解决了DUB模块如何通过与H2 A/H2 B二聚体或与核小体DNA的特异性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进行定量研究，在一个确定的，均匀的系统。酶动力学、结构测定和体内测定的测定将用于鉴定佐贺DUB模块和负责适当底物识别的核小体中的决定簇。从这些研究中出现的发现将与人类佐贺复合物直接相关，其亚基和组成与酵母复合物高度相似。由于人类DUB模块蛋白的缺陷直接涉及脊髓小脑共济失调7型，而人类Ubp 8同源物Usp 22是一种癌症干细胞标志物，因此我们的研究将为DUB功能障碍如何引起人类疾病提供直接见解。

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.