Capture of Ubiquitin Conjugation and Deconjugation Enzyme Substrates

描述（由申请人提供）：本申请涉及广泛的挑战领域（06）使能技术和特定的挑战主题06-CA-102：癌症中的瞬时分子复合物。几乎所有的细胞过程，包括那些参与细胞生长，分化和凋亡，依赖于特定的和仔细调节的蛋白质-蛋白质缔合和解离反应。这些反应的失调是许多人类癌症的特征和常见原因。由于蛋白质复合物的相互作用往往是微弱和短暂的，关键复合物的鉴定和表征往往是非常困难或不成功的。这个问题在泛素结合（E3泛素连接酶）和去泛素化（DUB）酶中表现得最为明显。由一个或多个76-氨基酸泛素分子对蛋白质进行共价修饰是所有真核生物中调节细胞内蛋白质水解的主要途径，因此它负责控制许多关键调节蛋白。此外，取决于特定的单或多泛素修饰，泛素化也可以导致其他命运。因此，泛素信号用于胞吞作用和蛋白质运输、转录激活、激酶激活级联和染色质重塑。在人类细胞中表达的数百种不同的E3和DUB酶中，已知的生理底物只有少数。这些酶底物复合物的瞬时性质和低丰度是大多数通过双杂交测定或基于亲和力的下拉实验鉴定底物的尝试失败的主要原因之一。该提议的目标是用称为同源复合物生物素标记（“CBiT”）的新方法来克服这些限制。在目的1中，修饰的生物素连接酶和生物素受体肽构建体将被设计为仅当这两种组分通过酶（E3或DUB）与其底物的相互作用而结合在一起时才允许生物素化。通过使生物素受体肽与泛素融合，即使是体内的瞬时E.S复合物也将经历生物素化，并且仅泛素缀合物将被标记。因此，真正的底物应该是高度富集后，亲和分离的生物素化的蛋白质，并服从质谱鉴定。将在酵母中用已知的E3.底物和DUB.底物对体内测试CBiT策略。在目标2中，将优化用于实施CBiT的细胞系和DNA载体，以有效应用于人类E3和DUB;在CBiT方法的测试中将使用与人类癌症和其他疾病有关的几种E3和DUB。 公共卫生相关性：蛋白质复合物的异常形成或稳定性是许多人类癌症和其他疾病的特征。由于蛋白质复合物的基础相互作用往往是微弱和短暂的，试图确定和表征关键蛋白质相互作用往往是不成功的或非常困难的。为了克服这个问题，一种新的方法称为同源复合物生物素标记（“CBiT”）将被开发和应用于复合物，调节泛素依赖的信号转导。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topic 06-CA-102: Transient Molecular Complexes in Cancer. Virtually all cellular processes, including those involved in cell growth, differentiation, and apoptosis, rely on specific and carefully regulated protein-protein association and dissociation reactions. Dysregulation of these reactions is characteristic and frequently a cause of many human cancers. Because the interactions that underlie protein complexes are often weak and transient, the identification and characterization of critical complexes often has been extremely difficult or unsuccessful. Nowhere has this problem been more evident than with ubiquitin conjugation (E3 ubiquitin ligase) and deubiquitination (DUB) enzymes. Covalent modification of proteins by one or more molecules of the 76-amino acid ubiquitin is the major route of regulated intracellular proteolysis in all eukaryotes, and as such it is responsible for the control of numerous key regulatory proteins. Additionally, depending upon the specific mono or polyubiquitin modification, ubiquitination also can lead to other fates. Thus, ubiquitin signals are used in endocytosis and protein trafficking, transcription activation, kinase activation cascades, and chromatin remodeling. Of the many hundreds of different E3 and DUB enzymes expressed in human cells, physiological substrates are known for only a handful. The transient nature and low-abundance of these enzyme.substrate complexes are among the principal reasons that most attempts to identify substrates by two- hybrid assays or affinity-based pulldown experiments have failed. The goal of this proposal is to overcome these limitations with a new approach termed cognate-complex biotin tagging ("CBiT"). In Aim 1, modified biotin ligase and biotin acceptor peptide constructs will be designed to allow biotinylation only when these two components are brought together by interaction of an enzyme (E3 or DUB) with its substrate. By having the biotin acceptor peptide fused to ubiquitin, even transient E.S complexes in vivo will be subject to biotinylation, and only ubiquitin conjugates will be tagged. As a result, bona fide substrates should be highly enriched after affinity-isolation of biotinylated proteins and amenable to identification by mass spectrometry. The CBiT strategy will be tested in vivo in yeast with known E3.substrate and DUB.substrate pairs. In Aim 2, cell lines and DNA vectors to implement CBiT will be optimized for efficient application with human E3s and DUBs; several E3s and DUBs that have been implicated in human cancers and other diseases will be used in tests of the CBiT approach. PUBLIC HEALTH RELEVANCE: Aberrant formation or stability of protein complexes is characteristic of many human cancers and other diseases. Because the interactions that underlie protein complexes are often weak and transient, attempts to identify and characterize critical protein interactions frequently are unsuccessful or extremely difficult. To overcome this problem, a new approach termed cognate-complex biotin tagging ("CBiT") will be developed and applied to complexes that regulate ubiquitin-dependent signaling.