Comprehensive proteomic profiling of the ubiquitin pathway in cancer

描述(申请人提供)：蛋白质修饰的泛素系统渗透到生物学的各个领域，与最常见的人类疾病密切相关，包括癌症、神经退行性变、炎症和病毒感染。泛素系统由大约50个E2和数百个E3组成，是真核生物中最具拜占庭性质的酶系统之一。虽然E1-E2-E3酶级联的基本原理现在已经确立，但泛素化酶的生物学功能和调控在很大程度上仍然是一个谜，主要是因为我们无法快速和全面地将泛素化酶与其细胞底物相匹配。为了解决这一先于泛素领域的困境，提出了一种新的基于活性的蛋白质捕获和图谱技术，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特定的方式无偏见地识别泛素化底物。(2)尽管结构信息已被证明无法将~50个E2与其数百个合作的E3相匹配，但SPASS方法提供了这一能力。(3)最后，对于大多数底物来说，修饰的特定赖氨酸残基是未知的，SPASS底物捕获和鉴定方法同样解决了这一缺点。在本项目中，SPASS将应用于人类前列腺癌同基因肿瘤发生和发展模型中~15个E2的分析。主要目标是确定这些E2的底物和协同作用的E3酶，精确定位底物赖氨酸的修饰，并量化底物利用的差异，随着前列腺细胞从永生化表型发展到致瘤表型。主要成果将是一个全面的平台和公开可用的数据集，将为癌症研究人员和临床医生提供一种新的资源，将基因组和蛋白质数据与癌症进展联系起来。 与公共卫生相关：泛素化酶(E2和E3)在细胞调节中的重要作用已将它们直接置于人类流行疾病，特别是癌症的中心。因此，几家制药公司已经在开发在治疗环境中调节泛素系统活动的策略。全面了解由泛素化酶控制的确切底物和途径，如本申请中建议的研究将为充分利用这种努力的潜力奠定基础。

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.