New deubiquitinating enzymes, poly-ubiquitin substrates and assays.

描述(由申请人提供)：脱泛素酶在泛素-蛋白酶体途径(UPP)中起着关键的调节作用。尽管它们与人类疾病相关，并受到制药商作为治疗开发目标的兴趣，但这种不同类别的酶还没有得到充分的研究，也缺乏详细的生化数据。高纯度的DUB酶和用于灵敏和均质分析的合适底物目前还没有商业化。在第一阶段，我们制备了新的泛素(Ub)蛋白和多泛素链，它们是成功的配音底物。该项目取得了成功，并产生了一套全面的工具，范围广泛，促进了新的DUBS和其他UPP组成部分的确定和特征。在这个提案中，我们在这一进展的基础上，重点关注两个主要主题：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DUBS以扩大我们目前可获得的商业酶的范围；ii)针对第一阶段生产的Ub底物和链测试整套DUB；以及iii)使用对Ub链的新修改来开发一种连续的基于面粉的DUB蛋白分解检测系统。我们将产生商业上可用的最全面的这类蛋白质小组。我们将使用我们现有的第一阶段生成的底物来表征这些DUB，以了解它们的生物学，并可能阐明它们在细胞中的功能。第一阶段的多泛素链原型将是修饰链的起点，创建一种通用的DUB底物，该底物最适合于荧光共振能量转移(FRET)等基于荧光的分析形式。底物还将在赖氨酸位置48和63处包含相关的异肽键连接，以剖析复制连接的偏好。这些试剂将商业化，以进一步推进DUB在蛋白质分解代谢、信号转导、细胞周期、细胞凋亡和神经变性方面的研究和药物开发。 公共卫生相关性：SBIR研究项目旨在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。具体地说，它与人类蛋白质“泛素”的分子生物化学有关，泛素在癌症、炎症、感染和神经退化等多种疾病状态中发挥作用。该项目产生的研究数据和有用的试剂盒产品将使泛素途径对各种人类疾病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.