Comprehensive proteomic profiling of the ubiquitin pathway in cancer

描述（由申请人提供）：蛋白质修饰的泛素系统渗透到生物学的每个领域，并与最普遍的人类疾病密切相关，包括癌症、神经变性、炎症和病毒感染。泛素系统包含约50个E2和估计数百个E3，是真核生物中最复杂的酶系统之一。虽然E1-E2-E3酶级联的基本原理现在已经很好地建立，但是泛素化酶的生物学功能和调节在很大程度上仍然是一个谜，主要是由于我们无法快速和全面地将泛素化酶与它们的细胞底物相匹配。作为解决这一问题的一种方法，提出了一种基于活性的蛋白质捕获和分析技术，称为SPASS（固相分析泛素化底物筛选）。SPASS同时解决了三个当代挑战：（1）它允许以E2特异性方式无偏见地鉴定泛素化底物。(2)虽然结构信息已被证明无法匹配的约50 E2与他们的数百个合作E3，SPASS方法提供了这种能力。(3)最后，修饰的特定赖氨酸残基对于大多数底物是未知的，这一缺点同样通过SPASS底物捕获和鉴定方法来解决。在本项目中，SPASS将应用于人类前列腺癌的同源肿瘤起始和进展模型中的~15个E2的分析。主要目标是确定这些E2的底物和协同E3酶，以查明被修饰的底物赖氨酸，并量化前列腺细胞从永生化到致瘤表型进展时底物利用的差异。主要交付成果将是一个综合平台和公开可用的数据集，为癌症研究人员和临床医生提供一种新的资源，将基因组和蛋白质组数据与癌症进展联系起来。 公共卫生相关性：泛素化酶（E2和E3）在细胞调节中的重要参与使其成为人类流行疾病，特别是癌症的中心。因此，一些制药公司已经在开发用于在治疗背景下调节泛素系统活性的策略。全面了解由泛素化酶控制的确切底物和途径，如本申请中提出的研究所能实现的，将为利用这些努力的全部潜力奠定基础。

DESCRIPTION (provided by applicant): The ubiquitin system of protein modification permeates every field in biology and is intimately linked to the most prevalent human diseases, including cancer, neurodegeneration, inflammation, and viral infections. With ~50 E2s and an estimated several hundred E3s, the ubiquitin system constitutes one of the most byzantine enzyme systems in eukaryotes. Whereas the basic principle of the E1-E2-E3 enzymatic cascade is now well established, the biological functions and regulation of ubiquitylation enzymes remain largely a mystery, mainly due to our inability to rapidly and comprehensively match ubiquitylation enzymes with their cellular substrates. As a solution to this predicament, which forestalls the entire ubiquitin field, a new activity-based protein capture and profiling technology, named SPASS (Solid Phase Assay for Ubiquitylation Substrate Screening) is proposed. SPASS simultaneously addresses three contemporary challenges: (1) It allows the unbiased identification of ubiquitylation substrates in an E2-specific manner. (2) Whereas structural information has proven unable to match the ~50 E2 with their hundreds of cooperating E3s, SPASS methodology provides this capability. (3) Lastly, the specific lysine residues that are modified are unknown for most substrates, a shortcoming that is equally addressed by the SPASS substrate capture and identification methodology. In the present project, SPASS will be applied to the profiling of ~15 E2s in a syngeneic tumor initiation and progression model of human prostate cancer. The main goal is to identify the substrates and cooperating E3 enzymes of these E2s, to pinpoint the substrate lysines that are modified, and to quantify differences in substrate utilization as prostate cells progress from an immortalized to a tumorigenic phenotype. The main deliverables will be a comprehensive platform and publicly available datasets that will provide cancer researchers and clinicians with a novel resource to link genomic and proteomic data with cancer progression. PUBLIC HEALTH RELEVANCE: The essential involvements of ubiquitylation enzymes (E2 and E3) in cellular regulation have placed them squarely at the center of prevalent human diseases, in particular cancer. Consequently, several pharmaceutical companies are already developing strategies for modulating ubiquitin system activity in a therapeutic context. A comprehensive understanding of the exact substrates and pathways controlled by ubiquitylation enzymes such as enabled by the studies proposed in this application will lay the foundation for harnessing the full potential of such efforts.