Revealing the regulatory mechanisms of endosomal cargo transporters

揭示内体货物转运蛋白的调控机制

• 批准号: 2337495
• 负责人: Daniel Capelluto
• 金额: $ 84.1万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2337495&HistoricalAwards=false
• 关键词: Revealing; regulatory; mechanisms; endosomal; cargo

This project will investigate the regulation of endosomal protein transporters in physiological and pathological scenarios. The poorly understood regulatory mechanisms of these transporters pose a significant bottleneck in the field. Cells modify their membrane protein receptors, as signals for internalization. These modified receptors, referred to as cargo, are delivered to endosomes, where the endosomal protein transporters recognize and guide the cargo for degradation. During bacterial infections, relocation of the endosomal protein transporters leads to bacterial survival in the host cell’s adverse environment. The Capelluto team will study regulatory mechanisms of this relocation using biophysics, structural biology, cell biology, and computational biology tools. The project will include recruiting senior undergraduate students from a nearby, historically black college, Bennett College through a new program, AccelerateSTEM. This program will offer a two-year training commitment at Virginia Tech, ending in an accelerated M.S. degree. The goal of AccelerateSTEM is to equip students with the training and resources needed to enhance their competitiveness in academic and nonacademic programs. The project aims to determine the molecular mechanisms governing the regulation of the endosomal cargo transporter TOM1. TOM1 binds ubiquitinated cargo through two domains, VHS and GAT. A highly conserved DXXLL sorting motif, located downstream of the TOM1 VHS domain, may be required for ubiquitinated cargo trafficking. Interestingly, the TOM1 DXXLL-containing region has been shown to be phosphorylated, suggesting a potential regulatory mechanism. The bacterium Shigella flexneri generates phosphatidylinositol 5-phosphate (PtdIns5P), promoting TOM1 recruitment to endosomes. This results in delayed endosome maturation, reduced protein turnover, and enhanced bacterial survival within host cells. Through a combination of isothermal titration calorimetry, NMR spectroscopy, molecular dynamic simulations, and cell-based experiments, the Capelluto team will test whether the DXXLL-containing region enhances the cargo trafficking function of TOM1 and if its phosphorylation plays a modulatory role. In the pathological scenario, the team will investigate, at the molecular and cellular levels, if both local acidification and PtdIns5P-dependent membrane binding impact TOM1’s cargo trafficking function during S. flexneri infection. These findings will advance our understanding of intracellular protein trafficking in both physiological and pathological contexts.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

本项目将研究生理和病理情况下内体蛋白转运体的调控。对这些转运体的调控机制了解甚少，这是该领域的一个重大瓶颈。细胞修饰它们的膜蛋白受体，作为内化的信号。这些被修饰的受体被称为“货物”，被递送到核内体，在那里核内体蛋白转运体识别并引导货物降解。在细菌感染期间，内体蛋白转运体的重新定位导致细菌在宿主细胞的不利环境中存活。Capelluto团队将使用生物物理学、结构生物学、细胞生物学和计算生物学工具研究这种迁移的调节机制。该项目将包括通过一个名为AccelerateSTEM的新项目，从附近的一所历史悠久的黑人学院贝内特学院（Bennett college）招募高年级本科生。该项目将在弗吉尼亚理工大学提供为期两年的培训承诺，以加速硕士学位结束。加速stem的目标是为学生提供所需的培训和资源，以提高他们在学术和非学术课程中的竞争力。该项目旨在确定控制内体货物转运体TOM1调节的分子机制。TOM1通过VHS和GAT两个结构域结合泛素化的货物。位于TOM1 VHS结构域下游的高度保守的DXXLL排序基序可能需要泛素化货物运输。有趣的是，含有TOM1 dxxll的区域已被证明是磷酸化的，这表明一个潜在的调控机制。福氏志贺氏菌产生5-磷酸磷脂酰肌醇（PtdIns5P），促进TOM1向核内体募集。这导致内核体成熟延迟，蛋白质周转减少，宿主细胞内细菌存活率提高。通过等温滴定量热法、核磁共振光谱法、分子动力学模拟和基于细胞的实验，Capelluto团队将测试含有dxxll的区域是否增强TOM1的货物运输功能，以及它的磷酸化是否起调节作用。在病理情况下，研究小组将在分子和细胞水平上研究局部酸化和ptdinsbp依赖的膜结合是否会影响flexneri感染期间TOM1的货物运输功能。这些发现将促进我们对生理和病理背景下细胞内蛋白质运输的理解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。