ATP-UBIQUITIN-DEPENDENT PROTEOLYSIS

描述（由申请人提供）：通过泛素/26S蛋白酶体途径进行的靶向蛋白质降解代表了真核细胞调节的基本策略，其中通过在26S蛋白酶体识别的赖氨酸和α-氨基上组装特定的多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途径的层次结构已经进化，以满足精确时空调节背景下全局特异性的相互冲突的需求。这些途径的异常靶向揭示了越来越多与细胞转化、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。当前资助期间的进展已经确定了有助于蛋白质相互作用的序列基序和特征，这些相互作用定义了选定的底物特异性、单个靶向途径的同源成分的识别以及组装多泛素降解信号的机制。在下一个资助期间扩展和利用这些发现的拟议研究包括四个具体目标。具体目标 1 将基于钼蝶呤合酶相关 MoeB-MoaD 异二聚体的结构序列建模，使用点诱变和动力学分析来测试有关泛素激活酶 (E1) 假定活性位点残基的具体预测。具体目标 2 将严格使用动力学和平衡研究来测试主要研究者最近的 V(max) 模型对 N 端规则特异性的预测，方法是使用氨基末端可以进行基因操作的已定义的二氢叶酸还原酶模型底物。具体目标 3 将使用动力学方法来检查 Mdm2 和 MdmX 催化的自动泛素化和 p53 缀合的机制、这些连接酶形成的非规范多泛素降解信号的连接特异性，以及将主要研究者最近的多泛素链形成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 将检查哺乳动物特异性泛素结合酶 E2(epf) 在 aCPI/hnRNP-EI/PCBP1（负责发育编程翻译沉默的 3'-UTR polyC(U) mRNA 结合蛋白）的靶向降解中的作用。总体而言，拟议的研究旨在应用传统的生化和遗传学方法，在分子水平上详细检查泛素依赖性 26S 蛋白酶体内的关键过程。

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.