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）中起关键的调节作用。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，但这类多样化的酶尚未得到充分研究，并且缺乏详细的生化数据。高度纯化的DUB酶和用于灵敏和均质测定的适当底物目前还没有市售。在第一阶段，我们制备了新的泛素（Ub）蛋白和多泛素链，它们是成功的泛素底物。这个项目是成功的，并产生了一个广泛的工具集，促进了新的dub和其他UPP组件的识别和表征。在本提案中，我们建立在这一进展和关注两个主要主题：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dub，以扩大我们目前的市售酶范围；ii)对照第一阶段生产的Ub底物和链对整套dub进行测试；iii)对Ub链进行新的修饰，开发基于面粉的DUB蛋白水解连续检测系统。我们将生成最全面的商业上可用的此类蛋白质面板。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特性，并可能阐明它们在细胞中的功能。I期多泛素链原型将是修饰链的起点，创建通用DUB底物，最适合荧光共振能量转移（FRET）等基于荧光的分析格式。底物还将在赖氨酸位置48和63处包含相关的异肽键，以便剖析DUB键的偏好。这些试剂将商业化，以进一步推进DUB研究和药物发现，包括蛋白质分解代谢、信号转导、细胞周期、细胞凋亡和神经变性。

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.