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：癌症中的瞬时分子复合体。几乎所有的细胞过程，包括那些参与细胞生长、分化和凋亡的过程，都依赖于特定的和仔细调节的蛋白质-蛋白质联合和解离反应。这些反应的失调是许多人类癌症的特征，而且经常是原因之一。由于构成蛋白质复合体的相互作用通常是微弱的和瞬时的，关键复合体的识别和表征通常是极其困难或不成功的。这个问题在泛素结合(E3泛素连接酶)和去泛素化(DUB)酶上表现得最为明显。由76个氨基酸组成的泛素的一个或多个分子对蛋白质进行共价修饰是所有真核生物细胞内蛋白降解的主要途径，因此它负责控制许多关键的调节蛋白。此外，根据特定的单泛素或多泛素修饰，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。在人类细胞表达的数百种不同的E3和DUB酶中，已知的生理底物只有一小部分。这些酶底物复合体的瞬变性质和低丰度是大多数通过双杂交分析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。这项提议的目标是通过一种名为同源复合体生物素标记(CBIT)的新方法来克服这些限制。在目标1中，修改的生物素连接酶和生物素受体多肽结构将被设计为只有当这两个成分通过酶(E3或DUB)与其底物相互作用而结合在一起时才允许生物素化。通过将生物素受体多肽与泛素融合，即使是体内短暂的E.S复合体也将受到生物素化的影响，只有泛素偶联物将被标记。因此，亲和分离生物素化蛋白质后，真正的底物应该高度丰富，并易于通过质谱学进行鉴定。CBIT策略将在具有已知E3底物和DUB底物对的酵母中进行体内测试。在目标2中，将优化用于实施CBIT的细胞系和DNA载体，以便有效地应用于人类E3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。 与公共卫生相关：蛋白质复合体的异常形成或稳定性是许多人类癌症和其他疾病的特征。由于构成蛋白质复合体的相互作用通常是微弱的和短暂的，因此经常尝试识别和表征关键的蛋白质相互作用是不成功的或极其困难的。为了解决这一问题，将开发一种名为同源复合体生物素标记(CBIT)的新方法，并将其应用于调节泛素依赖信号的复合体。

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.