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.

该项目将研究内体蛋白转运蛋白在生理和病理情况下的调节，而人们对这些转运蛋白的调节机制知之甚少，这在细胞修饰其膜蛋白受体（称为内化信号）方面构成了重大瓶颈。作为货物，被递送到内体，内体蛋白转运蛋白识别并引导货物降解。在细菌感染期间，内体蛋白转运蛋白的重新定位导致细菌在宿主细胞的不利环境中生存。卡佩卢托团队将利用生物物理学、结构生物学、细胞生物学和计算生物学工具来研究这种搬迁的调控机制，该项目将包括通过一个新项目 AccelerateSTEM 从附近历史悠久的黑人学院贝内特学院招募高年级本科生。 AccelerateSTEM 的目标是为学生提供增强学术和非学术课程竞争力所需的培训和资源。确定控制内体货物转运蛋白 TOM1 的分子机制 TOM1 通过 VHS 和 GAT 两个结构域结合泛素化货物，位于 TOM1 VHS 结构域下游的高度保守的 DXXLL 分选基序可能是泛素化货物运输所必需的。提示，含有 TOM1 DXXLL 的区域已被磷酸化，这表明福氏志贺氏菌存在潜在的调节机制。通过结合等温滴定量热法、核磁共振波谱法、分子动力学模拟和技术，生成磷脂酰肌醇 5-磷酸 (PtdIns5P)，促进 TOM1 募集到内体，从而延迟内体成熟、减少蛋白质周转并增强细菌存活率。基于细胞的实验，Capelluto团队将测试含有DXXLL的区域是否增强了TOM1的货物运输功能，以及它是否在病理情况下，研究小组将在分子和细胞水平上研究局部酸化和 PtdIns5P 依赖性膜结合是否会影响福氏链球菌感染期间 TOM1 的货物运输功能。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。