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泛素（Ub）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的polyUb类型，其不同之处在于链中的Ub-Ub键。每种形式的修饰被认为仅与功能结果的子集相关，这表明下游受体可以通过选择性结合来区分不同的polyUb拓扑结构。许多类型的泛素结合结构域（UBD）已被确定，但很少有人知道是连接特异性的，其中，其特异性的基础最近才开始被理解。我们已经描述了如何在蛋白质或蛋白质复合物中聚集两个或更多个UBD可以赋予与polyUb的高亲和力，连接选择性相互作用。这个模型，“连接特异性亲合力”，解释了K63连接的polyUb与参与DNA双链断裂修复灶组装的受体蛋白的特异性结合，它也有助于解释用去泛素化酶ataxin-3观察到的K48-polyUb结合偏好。在本提案中，我们开始沿着沿着两个主要方面扩展和应用我们的研究结果。首先，连接特异性亲合力的原理将被应用于工程化polyUb结合蛋白，其具有增强的选择性和亲和力，以及对新型polyUb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混合连接形式的polyUb。将使这些结合蛋白的版本特异性识别游离polyUb链的类型，所述游离polyUb链在NfkB活化和病毒诱导的先天免疫中被认为是关键信号。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UBD的特异性和功能。第二，一组模块化的，设计的UBD基序与不同的亲和力和连接特异性将被用来系统地探测连接特异性的作用polyUb受体功能。我们将重点关注具有三种不同功能的polyUb受体蛋白：人类Rap 80需要将BRCA 1修复复合物组装到DNA双链断裂的位点;共济失调蛋白-3是一种去泛素化酶，可能在蛋白质质量控制中发挥作用，并且是polyQ神经退行性疾病脊髓小脑共济失调3的病原体;以及酵母Rad 23，一种原型UBL-UBA蛋白，其将polyUb-蛋白缀合物带到26 S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连接特异性在这些过程中的重要性尚不清楚。我们将通过测试polyUb结合特性的系统性改变的影响来解决这个问题。这些实验将为人们普遍持有的观点提供首次实验测试，即（聚）Ub受体的连接选择性指导下游功能。

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.