Linkage-specific recognition of polyubiquitin

多聚泛素的连接特异性识别

• 批准号: 8479378
• 负责人: Robert Cohen
• 金额: $ 27.14万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8479378
• 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

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(申请人提供)：在所有真核生物中，许多通路和信号网络依赖于蛋白质泛素(Ub)聚合物的修饰来调节数千种蛋白质的水平、定位、相互作用或活性。已知的八种结构上不同的PolyUb类型因链中的Ub-Ub链接(S)而不同。每种形式的修饰被认为只与功能结果的一个子集相关，这表明下游受体可以通过选择性结合区分不同的PolyUb拓扑。许多类型的泛素结合域(UBD)已被确定，但很少有人已知是链接特异性的，其中，其特异性的基础直到最近才开始被理解。我们已经描述了如何将蛋白质或蛋白质复合体中的两个或更多UBD聚集在一起，从而实现与PolyUb的高亲和力、连接选择性的相互作用。这个模型，“链接特异性亲和力”，解释了K63连接的PolyUb与参与DNA双链断裂修复中心组装的受体蛋白的特异性结合，也有助于解释脱泛素酶ataxin-3观察到的K48-PolyUb结合偏好。在这项提案中，我们着手从两个主要方面扩展和应用我们的发现。首先，连接特异性亲和力的原理将被应用于设计具有增强的选择性和亲和力的PolyUb结合蛋白，并具有新形式的PolyUb的特异性。我们将开发高亲和力、高选择性的结合蛋白，识别K63、K48和K48/K63-PolyUb的混合连接形式。这些结合蛋白的不同版本将专门识别各种类型的游离PolyUb链，这些链被认为是NFkB激活和病毒诱导的先天性免疫的关键信号。作为这些蛋白质设计基础的策略也将被用来发现迄今尚未解决的UBD的特异性和功能。其次，我们将使用一组模块化的、设计的、具有不同亲和力和连锁特异性的UBD基序来系统地探讨连锁特异性在PolyUb受体功能中的作用。我们将关注具有三种非常不同功能的PolyUb受体蛋白质：人类Rap80是将BRCA1修复复合体组装到DNA双链断裂位点所必需的蛋白质；ataxin-3是一种可能在蛋白质质量控制中发挥作用的去泛素酶，它是多Q神经退行性疾病脊髓小脑型共济失调3的病原体；以及酵母RAD23，它是一种典型的UBL-UBA蛋白质，它将PolyUb-蛋白质结合物带到26S蛋白酶体进行降解。这些蛋白质的共同点是它们的功能需要PolyUb结合，但亲和力和Ub-Ub连锁特异性在这些过程中的重要性尚不清楚。我们将通过测试系统改变PolyUb结合属性的效果来解决这个问题。这些实验将为普遍持有的观点提供第一次实验测试，即(多)Ub受体的连接选择性指导下游功能。