Structure and Regulatory Mechanism of the Ubp8 Deubiquitinase Module

描述（由申请人提供）：Ubp 8去泛素化酶形成由Ubp 8、Sgf 73、Sgf 11和Sus 1亚基组成的四聚体复合物。我们有结构和生物化学证据表明，锌指结构域的Sgf 11和Sgf 73服务的双重功能，组织的催化核心的Ubp 8在一个酶活性构象，以及指导底物特异性对泛素化组蛋白H2 B（H2 B）。利用X射线晶体学和生物化学方法，我们将在分子和原子水平上剖析去泛素化酶（DUB）调节的机制。从靶蛋白中去除泛素对于基因转录、细胞周期进程和细胞分化等基本细胞过程是必不可少的。因此，DUB的突变、缺陷和操纵已经在几种人类疾病中被描述，即癌症、进行性神经变性和致病性病毒感染。具体而言，Ubp 8（人类USP 22）已被报道是参与癌症预后不良的癌症干细胞的11个基因签名的成员。Sgf 73（人类共济失调蛋白-7）与遗传性进行性脊髓小脑共济失调有关。通过阐明这种蛋白质复合物的结构和调控机制，我们将为未来的研究提供一个框架，旨在操纵DUB酶。这些方法将包括但不限于药理学分子筛选、基因治疗和新化合物的定向设计以激活或抑制DUB活性。我们建议，通过有效和特异性地控制去泛素化酶的酶活性，其失调的病理影响可能会减轻。使用X射线晶体学，我们可以确定蛋白质的精确三维结构。这种结构知识可以作为理解蛋白质如何被细胞的自然机制激活和抑制的模型。有了这些知识，我们就可以做出明智的决定，如何测试和设计激活或抑制这些蛋白质的疗法。具体来说，我们对Ubp 8复合物的研究将指导我们对一类酶的理解，这些酶被称为去泛素化酶，几乎参与了所有的生物学过程，在癌症进展、神经退行性变和微生物感染中起着重要作用。通过了解它们的三维结构，以及这种蛋白质复合物的各个部分如何对其整体功能做出贡献，我们希望指导旨在消除由去泛素化酶失调引起的疾病的研究。 公共卫生相关性：使用X射线晶体学，我们可以确定蛋白质的确切三维结构。这种结构知识可以作为理解蛋白质如何被细胞的自然机制激活和抑制的模型。有了这些知识，我们就可以做出明智的决定，如何测试和设计激活或抑制这些蛋白质的疗法。具体来说，我们对Ubp 8复合物的研究将指导我们对一类酶的理解，这些酶被称为去泛素化酶，几乎参与了所有的生物学过程，在癌症进展、神经退行性变和微生物感染中起着重要作用。通过了解UBp 8复合物的三维结构，以及这种蛋白质复合物的各个部分如何对其整体功能做出贡献，我们希望指导研究消除由去泛素化酶的错误调节引起的疾病。

DESCRIPTION (provided by applicant): The Ubp8 deubiquitinase forms a tetrameric complex consisting of Ubp8, Sgf73, Sgf11, and Sus1 subunits. We have structural and biochemical evidence that the zinc finger domains of Sgf11 and Sgf73 serve dual functions of organizing the catalytic core of Ubp8 in an enzymatically competent conformation as well as directing substrate specificity toward ubiquitylated histone H2B (H2B). Using X-ray crystallographic and biochemical approaches, we will dissect, at the molecular and atomic levels, the mechanisms of deubiquitinase (DUB) regulation. Removal of ubiquitin from target proteins is essential for cellular processes as fundamental as gene transcription, cell cycle progression, and cell differentiation. As such, mutations, deficiencies, and manipulation of DUBs have been described in several human diseases, namely cancer, progressive neurodegeneration, and pathogenic virus infection. Specifically, Ubp8 (USP22 in humans) has been reported to be a member of an 11-gene signature of cancer stem cells involved in poor cancer outcome prognosis. Sgf73 (Ataxin-7 in humans) has been implicated in an inherited, progressive spinocerebellar ataxia. By elucidating the architecture and regulatory mechanism of this protein complex, we will provide a framework for future research aimed at manipulation of DUB enzymes. These approaches would include, but would not be limited to pharmacological molecule screening, gene therapy, and directed design of novel compounds to activate or inhibit DUB activity. We propose that by potently and specifically controlling the enzymatic activity of deubiquitinases, the pathologic effects of their misregulation may be alleviated. Using X-ray crystallography, we can determine the exact three-dimensional structure of proteins. This structural knowledge can serve as a model to understand how proteins are activated and inhibited by the cell's natural machinery. With this knowledge in hand, we can make informed decisions about how to test and design therapies to activate or inhibit these proteins. Specifically, our study of the Ubp8 complex will guide our understanding of a class of enzymes, known as deubiquitinases, shown to be involved in virtually all biological processes, with fundamental roles in cancer progression, neurodegeneration, and microbial infection. By understanding their three-dimensional structure, and how the parts of this protein complex contribute to its function as a whole, we hope to guide studies aimed at eliminating ailments caused by misregulation of deubiquitinases. PUBLIC HEALTH RELEVANCE: Using X-ray crystallography, we can determine the exact three-dimensional structure of proteins. This structural knowledge can serve as a model to understand how proteins are activated and inhibited by the cell's natural machinery. With this knowledge in hand, we can make informed decisions about how to test and design therapies to activate or inhibit these proteins. Specifically, our study of the Ubp8 complex will guide our understanding of a class of enzymes, known as deubiquitinases, shown to be involved in virtually all biological processes, with fundamental roles in cancer progression, neurodegeneration, and microbial infection. By understanding the three-dimensional structure of the UBp8 complex, and how the individual parts of this protein complex contribute to its function as a whole, we hope to guide studies to eliminate ailments caused by misregulation of deubiquitinases.