ATP-UBIQUITIN-DEPENDENT PROTEOLYSIS

描述（由申请人提供）：通过泛素/26 S蛋白酶体途径的靶向蛋白质降解代表了真核细胞调控的基本策略，其中通过在26 S蛋白酶体识别的赖氨酸和α-氨基上组装特异性聚泛素降解信号来标记蛋白质的降解。在一个共同的酶机制内组织的平行泛素靶向通路的层次结构已经发展到满足精确的空间和时间调节的背景下的全球特异性的相互冲突的需求。这些通路的异常靶向解释了越来越多的与细胞转化、肿瘤发生、病毒感染、高血压和出生缺陷相关的发育和医学异常。在目前的资助期内取得的进展已经确定了序列基序和功能，有助于蛋白质的相互作用，定义选定的底物特异性，识别个别靶向途径的同源成分，以及组装聚泛素降解信号的机制。在下一个资助期内扩展和利用这些研究结果的拟议研究包括四个具体目标。具体目标1将使用点诱变和动力学分析来测试关于泛素激活酶（El）的推定活性位点残基的具体预测，其基于对多巴胺合酶的相关MoeB-MoaD异源二聚体的结构-序列建模。具体目标2将严格使用动力学和平衡研究，以测试主要研究者最近的V（最大）模型的预测N-末端规则的特异性，通过使用一个定义的二氢叶酸还原酶模型底物，其氨基末端可以遗传操作。具体目标3将使用动力学方法来检查Mdm 2和MdmX催化的autoubiquitination和p53共轭的机制，这些连接酶形成的非典型的多聚泛素降解信号的连接特异性，以及将主要研究者最近的多聚泛素链形成的双位点模型外推到环H2连接酶的一般功能背景的有效性。具体目标4将检查拟南芥特异性泛素缀合酶E2（epf）在aCPI/hnRNP-EI/PCBP 1的靶向降解中的作用，aCPI/hnRNP-EI/PCBP 1是负责发育程序性翻译沉默的3 '-UTR polyC（U）mRNA结合蛋白。总的来说，拟议的研究旨在将常规的生物化学和遗传学方法应用于在分子水平上对泛素依赖性26 S蛋白酶体靶向的关键过程进行详细检查。

DESCRIPTION (provided by applicant): Targeted protein degradation through the ubiquitin/26S proteasome pathway represents a fundamental strategy for eukaryotic cellular regulation in which proteins are marked for degradation by assembly of specific polyubiquitin degradation signals on lysine and a-amino groups that are recognized by the 26S proteasome. A hierarchy of parallel ubiquitin targeting pathways organized within a common enzymatic mechanism has evolved to meet the conflicting demands of global specificity within the context of precise spatial and temporal regulation. Aberrant targeting by these pathways explicates a growing list of developmental and medical abnormalities related to cell transformation, tumorigenesis, viral infection, hypertension, and birth defects. Progress during the current funding period has identified sequence motifs and features contributing to the protein interactions that define selected substrate specificity, recognition of cognate components of individual targeting pathways, and the mechanism for assembling the polyubiquitin degradation signals. Proposed studies to extend and exploit these findings during the next funding period comprise four Specific Aims. Specific Aim 1 will use point mutagenesis and kinetic analysis to test specific predictions regarding putative active site residues of ubiquitin activating enzyme (El), based on structure-sequence modeling of the related MoeB-MoaD heterodimer of molybdopterin synthase. Specific Aim 2 will use kinetic and equilibrium studies rigorously to test predictions of the principal investigator's recent V(max) model for N-end rule specificity by using a defined dihydrofolate reductase model substrate whose amino terminus can be genetically manipulated. Specific Aim 3 will use kinetic methods to examine the mechanism of Mdm2- and MdmX-catalyzed autoubiquitination and p53 conjugation, the linkage specificity of the non-canonical polyubiquitin degradation signals formed by these ligases, and the validity of extrapolating the principal investigator's recent two-site model for polyubiquitin chain formation to a general functional context for Ring H2 ligases. Specific Aim 4 will examine the role of the mammalian-specific ubiquitin conjugating enzyme E2(epf) in the targeted degradation of aCPI/hnRNP-EI/PCBP1, the 3'-UTR polyC(U) mRNA binding protein responsible for developmentally-programmed translational silencing. Overall, the proposed studies seek to apply conventional biochemical and genetic methods to the detailed examination of key processes within ubiquitin-dependent 26S proteasome targeting at the molecular level.