Comprehensive Analysis of Peptide Motif Binding In Vivo

体内肽基序结合的综合分析

• 批准号: 10707030
• 负责人: PETER M PRYCIAK
• 金额: $ 33.5万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707030
• 关键词: 3-Dimensional; Affinity; Agreement; Benchmarking; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Biology; Cell division; Cell physiology; Cells; Cyclin-Dependent Kinases; Cyclins; Data Set; Dependence; Disease; Docking; Drug Design; Enzymes; Event; Family; Goals; Growth; Health; Human; In Vitro; Knowledge; Left; Libraries; Link; Measures; Mediating; Methodology; Methods; Molecular; Monitor; Multiprotein Complexes; Neoplasms; Normal Cell; Pathology; Peptides; Phage Display; Pharmaceutical Preparations; Pharmacology; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Post-Translational Protein Processing; Protein Binding Domain; Proteins; Proteome; Proxy; Regulation; Role; Signaling Protein; Site; Specificity; Substrate Interaction; Surveys; Tertiary Protein Structure; Testing; Tissue Differentiation; Variant; Viral; Work; Yeasts; cell growth regulation; cost; design; experimental study; flexibility; high throughput screening; human pathogen; in vivo; inhibitor; meter; mutation screening; novel; preference; protein aminoacid sequence; protein folding; protein protein interaction; rapid technique; screening; tool; ubiquitin ligase; ubiquitin-protein ligase

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.

项目摘要/摘要PI/PD：Pryciak，Peter M. 细胞的正确功能取决于不同蛋白质之间的大量相互作用。 弱且短暂的相互作用在控制快速的分子事件中尤其重要 和动态。在许多情况下，这些瞬态相互作用是由三维折叠蛋白介导的 一个伴侣蛋白中的域与另一个伴侣中的短肽序列结合。这些肽 序列称为短线性基序或纤细的序列，在人类蛋白质中，有200多个不同的不同 纤细结合域和数百个例子的家庭。细长介导的相互作用至关重要 通过亚细胞定位，动态多蛋白质复合物的组装和底物识别的作用 翻译后修饰酶，例如激酶，磷酸酶，泛素连接酶等 已经对示例进行了深入研究，对于绝大多数纤细结合域而言，关键序列 控制其目标基序的识别的特征的定义很差。此外，超过300万个残留物 人类蛋白质组被预测为结构无序，因此可能包含许多尚未达到的 未发现的苗条。该提案旨在阐明细胞介导的相互作用和 填补当前的知识空白。实验将开发一种快速定量相对结合的方法 用于数千种变异肽基序序列的强度，以及对纤细序列的系统询问 控制他们的识别和效力的特征。该方法将使用细胞内功能测定法 可以为各种球状域定义细长的识别规则，为相对亲和力排名 大量的候选基序序列，甚至确定可以告知的竞争性抑制剂肽 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 效力和生化亲和力，以及纤细残留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是的约束亲和力的范围 通过该方法解决。另一个目标是将方法应用于大量细长的结合 域以表征其序列偏好并独立确认大量结合 在单独的筛选中鉴定出的肽。第三个目标是开发该方法的其他改编 将允许设计和优化基于肽的抑制剂和链接分子来连接 细胞内部不同的域。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，与正常细胞功能的基础机制有关 疾病状态，包括人类病原体劫持细长的结合域。