Capture of Ubiquitin Conjugation and Deconjugation Enzyme Substrates

泛素结合和解结合酶底物的捕获

基本信息

批准号：
7939805
负责人：
Robert Cohen
金额：
$ 35万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
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/7939805
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

项目摘要

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）：此申请涉及广泛的挑战领域（06）促成技术和特定挑战主题06-CA-102：癌症中的瞬时分子复合物。实际上，所有细胞过程，包括参与细胞生长，分化和凋亡的所有细胞过程，都依赖于特定和仔细调节的蛋白质 - 蛋白质蛋白缔合和解离反应。这些反应的失调是特征性的，并且通常是许多人类癌症的原因。由于蛋白质复合物构成的相互作用通常是弱且瞬时的，因此临界复合物的识别和表征通常非常困难或失败。没有比泛素结合（E3泛素连接酶）和去泛素化（DUB）酶更明显的问题。通过76-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，它负责控制众多关键调节蛋白。此外，根据特定的单声道或多泛素的修饰，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，激酶激活级联反应和染色质重塑。在人类细胞中表达的数百种不同的E3和配音酶中，生理底物仅以少数为名。这些酶的瞬时性质和低丰度。底层络合物是大多数尝试通过两种混合测定或基于亲和力的下拉实验识别底物的主要原因之一。该提案的目的是通过称为同类复合生物素标记的新方法来克服这些局限性（“ CBIT”）。在AIM 1中，修饰的生物素连接酶和生物素受体肽构建体才能设计出仅当通过酶（E3或DUB）与其底物的相互作用将这两个成分汇总在一起时，才允许生物素化。通过将生物素受体肽融合到泛素中，即使体内的瞬时E.s复合物也会受到生物素化的作用，并且只有泛素结合物被标记。结果，在生物素化蛋白亲和隔离后，应高度富集真正的底物，并且可以通过质谱法鉴定。 CBIT策略将在酵母中以已知的E3.sbrate和Dub.substrate对在体内进行测试。在AIM 2中，将优化实施CBIT的细胞系和DNA矢量，以有效地使用人E3和DUB。与人类癌和其他疾病有关的几个E3和配音将用于CBIT方法的测试。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。由于蛋白质复合物构成的相互作用通常是弱且瞬时的，因此经常识别和表征关键蛋白质相互作用的尝试不成功或极其困难。为了克服这个问题，将开发一种称为同源复合物生物素标记（“ 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。