Linkage-specific recognition of polyubiquitin

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。已知有8种结构不同的聚脲b，它们的不同之处在于链中的Ub-Ub连锁。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polyb拓扑结构。许多类型的泛素结合域（UBDs）已经被确定，但很少被认为是连锁特异性的，其中，其特异性的基础直到最近才开始被理解。我们已经描述了在一个蛋白质或蛋白质复合体内聚集两个或多个UBDs如何与聚脲b产生高亲和力、连接选择性的相互作用。这一“连接特异性亲和性”模型解释了DNA双链断裂时参与修复位点组装的受体蛋白对k63 -连接的聚脲的特异性结合，也有助于解释脱泛素化酶ataxin-3观察到的k48 -聚脲结合偏好。在这项建议中，我们着手在两个主要方面扩展和应用我们的研究结果。首先，链接特异性亲和性原理将应用于设计具有增强选择性和亲和力的聚脲b结合蛋白，并具有新形式聚脲b的新特异性。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-混合链接形式的polyb。这些结合蛋白的不同版本将特异性识别在NfkB激活和病毒诱导的先天免疫中作为关键信号的游离polyb链类型。这些蛋白质设计的基础策略也将用于发现迄今为止尚未解决的UBDs的特异性和功能。其次，将使用一组模块化设计的具有不同亲和力和连锁特异性的多聚脲受体基序，系统地探讨连锁特异性在多聚脲受体功能中的作用。我们将重点关注具有三种不同功能的polyb受体蛋白：人类Rap80需要将BRCA1修复复合体组装到DNA双链断裂的位点；ataxin-3是一种去泛素化酶，可能在蛋白质质量控制中起作用，是多发性神经退行性疾病脊髓小脑性共济失调3的致病因子；酵母Rad23是一种典型的UBL-UBA蛋白，它将polyb蛋白偶联物带到26S蛋白酶体上进行降解。这些蛋白的共同之处在于它们的功能需要与多聚脲结合，但亲和性和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聚脲结合特性的影响来解决这个问题。这些实验将为普遍持有的（多）Ub受体的连接选择性指导下游功能的观点提供第一个实验测试。

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.