Structure and Regulatory Mechanism of the Ubp8 Deubiquitinase Module

描述（由申请人提供）：Ubp8去泛素酶形成由Ubp8、Sgf73、Sgf11和Sus1亚基组成的四聚体复合物。我们有结构和生化证据表明，Sgf11和Sgf73的锌指结构域具有双重功能，即将Ubp8的催化核心组织成酶活性的构象，以及将底物特异性指向泛素化组蛋白H2B （H2B）。利用x射线晶体学和生化方法，我们将在分子和原子水平上解剖去泛素酶（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至关重要。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。具体来说，据报道，Ubp8（人类中的USP22）是参与癌症预后不良的癌症干细胞的11个基因标记的成员。Sgf73（人类Ataxin-7）与遗传性进行性脊髓小脑性共济失调有关。通过阐明该蛋白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。我们提出，通过有效和特异性地控制去泛素酶的酶活性，可以减轻其失调的病理效应。利用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一个模型来理解蛋白质是如何被细胞的自然机制激活和抑制的。有了这些知识，我们就可以在如何测试和设计激活或抑制这些蛋白质的疗法方面做出明智的决定。具体来说，我们对Ubp8复合物的研究将指导我们对一类酶的理解，即去泛素酶，它几乎参与了所有的生物过程，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体的部分如何促进其整体功能，我们希望指导旨在消除由去泛素酶失调引起的疾病的研究。

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.