Understanding the structural mechanism of spontaneous ubiquitin cargo clustering on the cell plasma membrane

了解细胞质膜上自发泛素货物聚集的结构机制

• 批准号: 10730734
• 负责人: Il-Hyung Lee
• 金额: $ 36.5万
• 依托单位: MONTCLAIR STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-06 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10730734
• 关键词: Atomic Force Microscopy; Binding; Binding Proteins; Biomimetic Materials; Capsid Proteins; Carrier Proteins; Cell membrane; Cell physiology; Cells; Cholesterol; Collaborations; Communication; Computer software; Crohn' s disease; Cues; Data; Data Analyses; Dependence; Disease; Engineering; Environment; Fluorescence; Fluorescence Microscopy; Goals; Golgi Apparatus; Health; Homeostasis; Image; Investigation; Learning; Length; Life; Lipid Bilayers; Lipids; Malignant Neoplasms; Mediating; Membrane; Membrane Microdomains; Membrane Proteins; Methods; Modeling; Molecular; Molecular Structure; Names; Non-Insulin-Dependent Diabetes Mellitus; Organelles; Organism; Organizational Change; Outcome; Pharmaceutical Preparations; Physical condensation; Physiological; Plasma Cells; Play; Polymers; Process; Proteins; RAD23B gene; Recruitment Activity; Research; Role; Sampling; Science; Scientist; Solid; Sorting; Structure; Students; Synapses; System; Tertiary Protein Structure; Testing; Therapeutic; Thermodynamics; Time; Transport Process; Ubiquitin; Underserved Population; Universities; Unsaturated Fats; Vesicle; Virus Diseases; Work; deep learning; design; environmental change; flexibility; insight; instrument; lipid transport; membrane model; membrane reconstitution; microscopic imaging; molecular modeling; nervous system disorder; neural; programs; protein structure; protein transport; receptor; reconstitution; recruit; response; skills; symposium; trafficking; undergraduate student; underserved students

Abstract The PI proposes to study the role of spontaneous Ubiquitin (UB) cargo clustering on the plasma membranes in vesicular trafficking using the model membrane reconstitution. Vesicular trafficking is a process of material transport by lipid vesicles between membrane bound organelles. The process is one of the very basic cellular functions and thus is involved in most life processes of higher organisms. However, its molecular mechanism is still poorly understood. Ubiquitylation, an enzymatic attachment of UB, is involved in many trafficking processes, but canonically, UB cargo has been considered only as a passive binder to the downstream trafficking proteins. Polymeric ubiquitylation of cargos and their multivalent interaction with binder proteins are very common in living cells leading to spontaneous protein condensate domain formation on the cell membranes. These cargo cluster- ing domains are also known to very closely interact with lipid raft domains. Understanding the spontaneous clustering of UB cargo as an active spatial organizer of the vesicular trafficking process can advance our current understanding of the vesicular trafficking mechanism by providing insight into the traditionally overlooked roles of UB cargo in the process. The main methods of this investigation are fluorescence microscopy (FM) and atomic force microscopy (AFM). Image data are analyzed by the deep-learning-assisted analysis software. The goals of this proposal are: (1) Identifying the structural organization of UB cargo clusters created by proteins with different structures; (2) Understanding the lipid composition dependence of the UB cluster organization. The PI places an emphasis on structural understanding of the cargo clustering domain formation in this proposal. FM is used for high throughput imaging of many samples with different protein structures to understand the structural cues that lead to clustering domain formation. AFM is used to study the structural organization of the clusters at the molecular level to obtain direct structural snapshots to test hypothetical models. The proposal will enhance the research environment at Montclair State University by synergistically enabling the use of the high-sensitivity AFM instrument the university recently obtained. Moreover, the proposal will address the current high demand of undergraduate students to participate in research while preparing them to advance to graduate programs in health-related sciences. In collaboration with the GS-LSAMP program, students of underserved population will be actively recruited. Students are expected to acquire experimental skills, learn data analysis, and present in conferences to meet and communicates with scientists in the field. 1

抽象的 PI建议研究自发泛素（UB）货物在质膜上的货物簇的作用 使用模型膜重建的囊泡贩运。囊泡贩运是一个材料的过程 通过膜结合细胞器之间的脂质囊泡运输。该过程是非常基本的细胞之一 功能，因此参与了较高生物体的大多数生命过程。但是，其分子机制是 仍然很了解。 UB的酶促附着的泛素化涉及许多贩运过程， 但是从规范上讲，UB货物仅被视为下游贩运蛋白的被动粘合剂。 嘉戈斯的聚合物泛素化及其与粘合剂蛋白的多价相互作用在生活中非常普遍 细胞导致细胞膜上自发蛋白凝结结构域形成。这些货物集群 - 已知ING域与脂质筏结构域非常紧密相互作用。了解自发 将UB货物作为囊泡贩运过程的活跃空间组织者聚集可以推动我们的当前 通过洞悉传统被忽视的角色，了解囊泡贩运机制 在此过程中的UB货物。该研究的主要方法是荧光显微镜（FM）和原子 力显微镜（AFM）。图像数据通过深度学习辅助分析软件进行分析。目标 该建议的内容是：（1）确定由蛋白质创建的UB货物集群的结构组织 不同的结构； （2）了解UB群集组织的脂质组成依赖性。 pi 在本提案中，强调对货物聚类域形成的结构理解。 FM是 用于许多具有不同蛋白质结构的样品的高吞吐量成像，以了解结构 导致聚类域形成的提示。 AFM用于研究集群的结构组织 分子水平获得直接的结构快照来测试假设模型。该提议将增强 蒙特克莱尔州立大学的研究环境通过协同使用高敏性来协同使用 AFM仪器大学最近获得了。此外，该提案将满足当前的高需求 本科生参加研究，同时准备他们晋升为研究生课程 与健康有关的科学。与GS-LSAMP计划合作，服务不足的学生将 积极招募。期望学生获得实验技能，学习数据分析并呈现 会议与该领域的科学家见面和沟通。 1