Comprehensive Analysis of Peptide Motif Binding In Vivo

项目总结/摘要PI/PD：Pryciak，Peter M. 细胞的正常功能取决于不同蛋白质之间的大量相互作用。 弱的和短暂的相互作用在控制快速的分子事件中特别重要 充满活力在许多情况下，这些瞬时相互作用是由三维折叠的蛋白质介导的 一个伴侣蛋白中的结构域与另一个伴侣中的短肽序列结合。这些肽 序列被称为短线性基序，或SLiM，在人类蛋白质中，有200多种不同的 SLiM结合结构域的家族和数百个实例。SLiM介导的相互作用是关键的 在亚细胞定位、动态多蛋白复合物的组装和底物识别中的作用， 翻译后修饰酶如激酶、磷酸酶、泛素连接酶等。 已经深入研究了一些例子，对于绝大多数SLiM结合结构域，关键序列 控制其目标基序识别的特征定义不清楚。此外，超过300万的残留物 预测人类蛋白质组在结构上是无序的，因此可能包含许多迄今为止 未发现的SLiM该提案旨在阐明SLiM介导的相互作用的分子基础， 填补现有知识空白。本实验将发展一种快速定量相对结合的方法 对数千种变体肽基序序列的强度，以及对SLiM序列的系统询问 控制其识别和效力的特征。该方法将使用细胞内功能测定， 可以为各种各样的球状域定义SLiM识别规则，为 大量的候选基序序列，甚至确定竞争性抑制剂肽，可以通知 药物设计一个目标是通过建立功能之间的相关性来验证方法 效力和生物化学亲和力，以及SLiM残基偏好对周围肽的依赖性 背景或力量。这些实验还将寻求扩大可以被应用的结合亲和力的范围。 通过该方法解决。另一个目标是将该方法应用于大量的SLiM结合。 结构域来表征它们的序列偏好，并独立地确认大量的结合 在单独的筛选中鉴定的肽。第三个目标将是开发该方法的其他适应性， 将允许设计和优化基于肽的抑制剂和连接分子， 细胞内不同的区域结合在一起。总的来说，这些研究将有助于我们对 蛋白质-蛋白质相互作用，与正常细胞功能以及 疾病状态，包括人类病原体劫持SLiM结合结构域。

Project Summary/Abstract PI/PD: Pryciak, Peter M. The proper function of cells depends on an enormous number of interactions between different proteins. Interactions that are weak and transient are especially important in controlling molecular events that are rapid and dynamic. In many cases these transient interactions are mediated by three-dimensionally folded protein domains in one partner protein that bind to short peptide sequences in the other partner. These peptide sequences are known as Short Linear Motifs, or SLiMs, and among human proteins there are over 200 distinct families of SLiM-binding domains and many hundreds of examples. SLiM-mediated interactions serve critical roles in subcellular localization, assembly of dynamic multi-protein complexes, and substrate recognition by post-translational modification enzymes such as kinases, phosphatases, ubiquitin ligases, etc. While some examples have been studied intensively, for the vast majority of SLiM-binding domains the key sequence features that govern recognition of their target motifs are poorly defined. Moreover, over 3 million residues of the human proteome are predicted to be structurally disordered and hence are likely to contain many as-yet undiscovered SLiMs. This proposal seeks to illuminate the molecular basis of SLiM-mediated interactions and fill current knowledge gaps. The experiments will develop a method for rapid quantification of relative binding strength for thousands of variant peptide motif sequences, and a systematic interrogation of SLiM sequence features that control their recognition and potency. The approach will use an intracellular functional assay that can define SLiM recognition rules for a wide variety of globular domains, provide a relative affinity ranking for large numbers of candidate motif sequences, and even identify competitive inhibitor peptides that can inform drug design. One goal will be to validate the methodology by establishing the correlation between functional potency and biochemical affinity, and the dependence of SLiM residue preferences on the surrounding peptide context or strength. These experiments will also seek to expand the range of binding affinities that can be resolved by the method. Another goal will be to apply the method toward a large number of SLiM-binding domains to characterize their sequence preferences and independently confirm large numbers of binding peptides identified in separate screens. A third goal will be to develop additional adaptations of the method that will allow for the design and optimization of peptide-based inhibitors and tethering molecules for linking together distinct domains inside cells. Overall, these studies will contribute to our general understanding of protein-protein interactions, with relevance to the mechanisms underlying normal cell function as well as disease states including the hijacking of SLiM-binding domains by human pathogens.