Capture of Ubiquitin Conjugation and Deconjugation Enzyme Substrates

泛素结合和解结合酶底物的捕获

• 批准号: 7825747
• 负责人: Robert Cohen
• 金额: $ 32.5万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7825747
• 关键词: Address; Affinity; Amino Acids; Apoptosis; Area; BARD1 gene; BRCA1 gene; Binding; Biological Assay; Biotin; Biotinylation; Cell Line; Cell physiology; Cells; Characteristics; Chimeric Proteins; Complex; DNA; Destinations; Deubiquitination; Disease; Dissociation; Endocytosis; Engineering; Enzyme Interaction; Enzymes; Eukaryota; Fluorescence; Fluorescence Polarization; Gene Expression; Goals; Histone H2B; Human; Label; Lead; Length; Ligands; Ligase; Link; Malignant Neoplasms; Mass Spectrum Analysis; Modification; Molecular; Mono-S; N-terminal; Nature; Pathway interactions; Peptides; Phosphotransferases; Physiological; Polyubiquitin; Post-Translational Protein Processing; Protein Binding Domain; Proteins; Proteolysis; RNA Interference; Reaction; Route; Signal Transduction; System; Technology; Testing; Transcriptional Activation; Transduction Gene; Two-Hybrid System Techniques; UBD protein; Ubiquitin; Ubiquitination; Variant; Yeasts; base; cell growth; chromatin remodeling; design; enzyme substrate; genetic regulatory protein; in vitro Assay; in vitro Model; in vivo; mutant; novel strategies; protein aminoacid sequence; protein complex; protein transport; public health relevance; research study; ubiquitin-protein ligase; vector

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.

描述（由申请人提供）：本申请涉及广泛的挑战领域 (06) 支持技术，以及具体的挑战主题 06-CA-102：癌症中的瞬时分子复合物。事实上，所有细胞过程，包括那些涉及细胞生长、分化和凋亡的细胞过程，都依赖于特定且精心调节的蛋白质-蛋白质结合和解离反应。这些反应的失调是许多人类癌症的特征，并且常常是许多人类癌症的原因。由于蛋白质复合物背后的相互作用通常很弱且短暂，因此关键复合物的识别和表征通常极其困难或不成功。这个问题在泛素结合酶（E3 泛素连接酶）和去泛素化 (DUB) 酶中表现得最为明显。 76 个氨基酸泛素的一个或多个分子对蛋白质的共价修饰是所有真核生物中受调节的细胞内蛋白水解的主要途径，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，泛素化也可能导致其他命运。因此，泛素信号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。在人体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二杂交测定或基于亲和力的下拉实验来鉴定底物的尝试失败的主要原因之一。该提案的目标是通过一种称为同源复合物生物素标签（“CBiT”）的新方法来克服这些限制。在目标 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个组件通过酶（E3 或 DUB）与其底物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 复合物也会发生生物素化，并且只有泛素缀合物才会被标记。因此，在亲和分离生物素化蛋白质后，真正的底物应该高度富集，并易于通过质谱法进行鉴定。 CBiT 策略将在酵母中用已知的 E3.底物和 DUB.底物对进行体内测试。在目标 2 中，将优化用于实施 CBiT 的细胞系和 DNA 载体，以实现与人类 E3 和 DUB 的有效应用；一些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。 公共健康相关性：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。由于蛋白质复合物背后的相互作用通常很弱且短暂，因此识别和表征关键蛋白质相互作用的尝试常常不成功或极其困难。为了克服这个问题，将开发一种称为同源复合物生物素标签（“CBiT”）的新方法，并将其应用于调节泛素依赖性信号传导的复合物。