Comprehensive Analysis of Peptide Motif Binding In Vivo

体内肽基序结合的综合分析

基本信息

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
项目类别：
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

项目摘要

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. 细胞的正常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对于控制快速的分子事件尤其重要和动态。在许多情况下，这些瞬时相互作用是由三维折叠蛋白介导的一个伙伴蛋白中的结构域与另一伙伴蛋白中的短肽序列结合。这些肽序列被称为短线性基序 (SLiM)，人类蛋白质中有 200 多种不同的序列 SLiM 结合域家族和数百个示例。 SLiM 介导的相互作用至关重要在亚细胞定位、动态多蛋白复合物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。实例已被深入研究，对于绝大多数 SLiM 结合域，关键序列控制目标图案识别的特征定义不明确。此外，超过300万个残留物人类蛋白质组预计在结构上是无序的，因此可能包含许多迄今尚未发现的蛋白质组。未被发现的 SLiM。该提案旨在阐明 SLiM 介导的相互作用的分子基础和填补当前的知识空白。该实验将开发一种快速定量相对结合的方法数以千计的变异肽基序序列的强度，以及 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球状结构域定义 SLiM 识别规则，提供相对亲和力排名大量候选基序序列，甚至可以识别竞争性抑制肽，从而提供信息药物设计。一个目标是通过建立功能之间的相关性来验证该方法。效力和生化亲和力，以及 SLiM 残基偏好对周围肽的依赖性背景或强度。这些实验还将寻求扩大可结合亲和力的范围方法解决。另一个目标是将该方法应用于大量 SLiM 绑定结构域来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。第三个目标是对该方法进行进一步的修改，将允许设计和优化基于肽的抑制剂和用于连接的束缚分子细胞内不同的域聚集在一起。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，与正常细胞功能的机制以及疾病状态，包括人类病原体劫持 SLiM 结合域。