New deubiquitinating enzymes, poly-ubiquitin substrates and assays.

描述（由申请人提供）：去泛素化酶在泛素-蛋白酶体途径（UPP）中起关键调节作用。尽管它们与人类疾病相关，并且制药公司将其作为治疗开发的靶点，但这类不同的酶研究不足，并且缺乏详细的生化数据。高度纯化的DUB酶和用于敏感和均相测定的适当底物目前还没有市售。在第一阶段，我们提出了新的泛素（Ub）蛋白和多聚泛素链，这是成功的DUB基板。该项目是成功的，并产生了一个全面的工具集，范围广泛，促进了新的DUB和其他UPP组件的识别和表征。在这个建议中，我们建立在这一进展，并专注于两个主要课题：新的去泛素化酶和新的泛素链底物。我们的具体目标是：i）开发新的DUB以扩大我们目前的市售酶范围; ii）针对阶段I中制备的Ub底物和链测试全套DUB;以及iii）使用Ub链上的新修饰以开发用于DUB蛋白水解的连续的基于面粉的测定系统。我们将产生最全面的面板，这种蛋白质是商业可用的。我们将使用我们现有的I期生成的底物来表征这些DUB，以了解它们的生物学，并可能揭示它们在细胞中的功能。I期多聚泛素链原型将是修饰链的起点，创建通用DUB底物，其对于基于荧光的测定形式（例如荧光共振能量转移（FRET））是最佳的。底物还将在赖氨酸位置48和63处含有相关的异肽键连接，以剖析DUB连接偏好。这些试剂将被商业化，以进一步推进DUB研究和药物发现的蛋白质催化剂，信号转导，细胞周期，细胞凋亡和神经变性。 公共卫生相关性：SBIR研究项目是支持生物制品和诊断实验室分析试剂盒的设计、开发和生产。具体而言，它涉及人蛋白质“泛素”的分子生物化学，所述蛋白质在许多疾病状态如癌症、炎症、感染和神经变性中起作用。该项目产生的研究数据和有用的试剂盒产品将使各种人类疾病的泛素途径的新研究和药物开发成为可能。

DESCRIPTION (provided by applicant): Deubiquitinating enzymes play critical regulatory roles in the ubiquitin-proteasome pathway (UPP). Despite their relevance to human disease and interest by pharma as targets for therapeutic development, this diverse class of enzymes is understudied and detailed biochemical data is lacking. Highly purified DUB enzymes and appropriate substrates for sensitive and homogeneous assays are not currently commercially available. In Phase I we made new ubiquitin (Ub) proteins and poly-ubiquitin chains which are successful DUB substrates. This project was successful and generated a comprehensive tool set with wide-ranging that facilitated the identification and characterization of new DUBs and other UPP components. In this proposal we build on that progress and focus on two major topics: new deubiquitinating enzymes and novel ubiquitin chain substrates. Our specific aims are to: i) develop new DUBs to expand our current range of commercially available enzymes; ii) test the complete set of DUBs against the Ub substrates and chains made in Phase I; and iii) use new modification on Ub chains to develop a continuous flour-based assay system for DUB proteolysis. We will generate the most comprehensive panel of such proteins that are commercially available. We will characterize these DUBs using our existing Phase I generated substrates to understand their biology and possibly shed light on their function in the cell. The Phase I poly-ubiquitin chain prototypes will be the starting point for modified chains, creating a universal DUB substrate that is optimal for fluorescent-based assay formats such as fluorescent resonance energy transfer (FRET). The substrate will also contain relevant isopeptide bond linkages at lysine positions 48 and 63, in order to dissect DUB linkage preferences. These reagents will be commercialized to further advance DUB research and drug discovery in protein catabolism, signal transduction, cell cycle, apoptosis, and neurodegeneration. PUBLIC HEALTH RELEVANCE: SBIR research project is to support the design, development, and production of biological products and diagnostic laboratory analysis kits. Specifically it relates to the molecular biochemistry of the human protein "ubiquitin" which has roles in numerous disease states such as cancer, inflammation, infection and neurodegeneration. The research data and useful kit products generated by this project will enable new research and drug development in the ubiquitin pathway for various human diseases.