Comprehensive Analysis of Peptide Motif Binding In Vivo

项目摘要/摘要PI/PD：Pryciak，Peter M. 细胞的正常功能依赖于不同蛋白质之间的大量相互作用。 弱而短暂的相互作用对于控制快速的分子事件尤为重要 动感十足。在许多情况下，这些瞬时相互作用是由三维折叠蛋白质介导的 一个伙伴蛋白中的结构域与另一个伙伴中的短肽序列结合。这些多肽 序列被称为短线状模体，或Slims，在人类蛋白质中有200多种不同的序列 细结合结构域的家族和数百个例子。超薄中介的交互服务至关重要 在亚细胞定位、组装动态多蛋白复合体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 已经对例子进行了深入的研究，对于绝大多数细小结合结构域的关键序列 管理其目标主题识别的特征定义不佳。此外，超过300万的残留物 人类蛋白质组被预测在结构上是无序的，因此很可能包含许多迄今 未被发现的苗条。这一提议试图阐明SLIM介导的相互作用的分子基础，并 填补当前的知识空白。这些实验将开发一种快速量化相对结合的方法 数千个变异的多肽基序序列的强度，以及对SLIM序列的系统询问 控制其识别和效力的功能。该方法将使用细胞内功能分析， 可以为各种球状域定义细小的识别规则，提供相对亲和力排名 大量的候选基序序列，甚至识别竞争抑制肽，这些抑制肽可以 药物设计。一个目标是通过建立功能之间的关联来验证该方法 效价和生化亲和力，以及细小残基偏好对周围多肽的依赖 背景或实力。这些实验还将寻求扩大结合亲和力的范围， 通过该方法进行解析。另一个目标是将该方法应用于大量的超薄装订 结构域来表征它们的序列偏好并独立地确认大量结合 在不同的屏幕中识别的多肽。第三个目标将是开发对该方法的其他适应 将允许设计和优化基于多肽的抑制剂和用于连接的系链分子 细胞内不同的结构域结合在一起。总体而言，这些研究将有助于我们对 蛋白质-蛋白质相互作用，与正常细胞功能的潜在机制以及 疾病状态，包括人类病原体劫持细小结合域。

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.