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，以及特定的挑战主题06- ca -102：癌症中的瞬态分子复合物。几乎所有的细胞过程，包括那些涉及细胞生长、分化和凋亡的过程，都依赖于特异性和精心调节的蛋白质-蛋白质结合和解离反应。这些反应的失调是特征性的，经常是许多人类癌症的原因。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极其困难或不成功的。这个问题在泛素偶联（E3泛素连接酶）和去泛素化（DUB）酶中表现得最为明显。一个或多个由76个氨基酸组成的泛素分子对蛋白质的共价修饰是所有真核生物细胞内蛋白水解调控的主要途径，因此它负责控制许多关键的调节蛋白。此外，根据特定的单或多泛素修饰，泛素化也可以导致其他命运。因此，泛素信号被用于内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。这些酶的短暂性和低丰度。底物配合物是大多数通过双杂交测定或基于亲和的下拉实验鉴定底物失败的主要原因之一。本提案的目标是通过一种称为同源复合体生物素标记（“CBiT”）的新方法来克服这些限制。在Aim 1中，修饰的生物素连接酶和生物素受体肽结构将被设计为只有当这两种成分通过酶（E3或DUB）与其底物的相互作用结合在一起时才允许生物素化。通过将生物素受体肽与泛素融合，即使是体内短暂的e - s复合物也会被生物素化，只有泛素缀合物才会被标记。因此，经过生物素化蛋白的亲和分离，真正的底物应该是高度富集的，并且可以通过质谱鉴定。CBiT策略将在已知E3的酵母菌体内进行测试。衬底和DUB。基质对。在目标2中，将优化用于实现CBiT的细胞系和DNA载体，以便有效地应用于人类E3s和dub；几种与人类癌症和其他疾病有关的e3和dub将用于CBiT方法的测试。

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.