Linkage-specific recognition of polyubiquitin

多聚泛素的连接特异性识别

基本信息

批准号：
8668081
负责人：
Robert Cohen
金额：
$ 28.11万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8668081
关键词：
26S proteasome Accounting Address Affinity Amino Acid Motifs Antiviral Agents Avidity BRCA1 gene Behavior Binding Binding Proteins Cells DNA Damage DNA Double Strand Break Defect Detection Deubiquitinating Enzyme Development Down-Regulation Engineering Enzyme Activation Eukaryota Exhibits Human In Vitro Individual Ligands Link Lysine MHC Class I Genes MJD1 protein Machado-Joseph Disease Malignant Neoplasms Modeling Modification Natural Immunity Neurodegenerative Disorders Physiology Polymers Polyubiquitin Process Property Proteasome Binding Proteins Quality Control Reading Reagent Repair Complex Role Signal Pathway Signal Transduction Signal Transduction Pathway Site Solutions Specificity Structure Testing Ubiquitin Ubiquitination Yeasts base design enzyme activity functional outcomes human disease in vivo inhibitor/antagonist multicatalytic endopeptidase complex novel polyglutamine preference protein complex protein degradation protein transport receptor receptor function repaired research study response tool

项目摘要

DESCRIPTION (provided by applicant): In all eukaryotes, numerous pathways and signaling networks depend upon modification by polymers of the protein ubiquitin (Ub) to regulate the levels, localization, interactions, or activities of thousands of proteins. Eight structurally distinct types of polyUb are known that differ by the Ub-Ub linkage(s) in the chain. Each form of the modification is thought to be associated with only a subset of functional outcomes, suggesting that downstream receptors can distinguish the different polyUb topologies through selective binding. Many types of ubiquitin binding domains (UBDs) have been identified, but few are known to be linkage-specific and, among those that are, the basis for their specificity has only recently begun to be understood. We have described how clustering two or more UBDs within a protein or protein complex can confer high-affinity, linkage-selective interactions with polyUb. This model, "linkage-specific avidity", explains the specific binding of K63-linked polyUb by receptor proteins involved in the assembly of repair foci at DNA double-strand breaks, and it also helps to account for the K48-polyUb binding preference observed with the deubiquitinating enzyme ataxin-3. In this proposal, we set out to extend and apply our findings along two major fronts. First, the principles of linkage-specific avidity will be applied to engineer polyUb binding proteins with enhanced selectivity and affinity, and with new specificities for novel forms of polyUb. We will develop high-affinity, high-selectivity binding proteins that recognize K63, K48, and K48/K63-mixed-linkage forms of polyUb. Versions of these binding proteins will be made to specifically recognize types of free polyUb chains that have been implicated as critical signals in NfkB activation and virally-induced innate immunity. Strategies that underlie the design of these proteins also will be used to discover the specificities and functions of UBDs that thus far have defied solution. Second, a set of modular, designed UBD motifs with varying affinities and linkage specificities will be used to systematically probe the role of linkage specificity in polyUb receptor function. We will focus on polyUb receptor proteins that have three very different functions: human Rap80 is needed to assemble the BRCA1 repair complex to sites of DNA double-strand breaks; ataxin-3 is a deubiquitinating enzyme with a likely role in protein quality-control and that is the causative agent of the polyQ neurodegenerative disease spinocerebellar ataxia 3; and the yeast Rad23, a prototypical UBL-UBA protein that brings polyUb-protein conjugates to the 26S proteasome for degradation. These proteins have in common that their functions require polyUb binding, but the importance of affinity and Ub-Ub linkage specificity in these processes is not known. We will address this issue by testing the effects of systematic alterations of polyUb binding properties. These experiments will provide the first experimental tests of the commonly-held view that the linkage-selectivity of (poly)Ub receptors directs downstream functions.

描述（由申请人提供）：在所有真核生物中，许多途径和信号网络都取决于蛋白质泛素（UB）的聚合物的修饰，以调节数千种蛋白质的水平，定位，相互作用或活性。已知八种结构上不同类型的polyub，这些类型与链中的UB-UB链接不同。修饰的每种形式都被认为仅与功能结果的子集有关，这表明下游受体可以通过选择性结合来区分不同的polyub拓扑。已经确定了许多类型的泛素结合结构域（UBD），但是很少有人被称为特定于链接的特定于链接，并且在那些是其特异性基础的基础中，直到最近才开始理解。我们已经描述了在蛋白质或蛋白质复合物中将两个或多个UBD聚类如何赋予高亲和力，连锁选择性相互作用与polyub的相互作用。该模型“链接特异性亲和力”解释了涉及DNA双链休息时涉及修复焦点的受体蛋白K63连接多核的特异性结合，并且还有助于考虑K48-Polyub结合偏好的观察与去泛素酶ataxin-3。在此提案中，我们着手扩展并应用我们的发现沿两个主要方面。首先，将链接特异性亲和力的原理应用于具有增强的选择性和亲和力的工程师Polyub结合蛋白，并具有新型Polyub形式的新特异性。我们将开发高亲和力，高选择性结合蛋白，这些蛋白可以识别polyub的K63，K48和K48/K63混合链接形式。这些结合蛋白的版本将被制成特异性识别的类型的游离多核链，这些链被认为是NFKB激活和病毒诱导的先天免疫的关键信号。这些蛋白质设计的基础的策略也将用于发现迄今为止违反解决方案的UBD的特异性和功能。其次，一组具有不同亲和力和连锁特异性的模块化的UBD图案将用于系统地探测链接特异性在Polyub受体功能中的作用。我们将专注于具有三个截然不同功能的多核受体蛋白：需要人RAP80来组装BRCA1修复复合物与DNA双链断裂部位的位置； ataxin-3是一种去泛素化酶，可能在蛋白质质量控制中起作用，也是polyq神经退行性疾病脊椎脑性共济失调3的病因。酵母Rad23是一种典型的UBL-UBA蛋白，将多蛋白蛋白结合到26S蛋白酶体中以降解。这些蛋白质通常是它们的功能需要polyub结合，但是在这些过程中，亲和力和UB-UB链接特异性的重要性尚不清楚。我们将通过测试Polyub结合特性的系统变化的影响来解决此问题。这些实验将提供第一个实验测试，即（Poly）UB受体的连锁选择性指导下游函数。