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.

该项目将调查在物理和病理方案中内体蛋白转运蛋白转运蛋白的调节。这些转运蛋白的调节机制知之甚少，在该领域构成了显着的瓶颈。细胞改变其膜蛋白受体，作为内在化的信号。这些修饰的受体（称为货物）被传递到内体蛋白转运蛋白转运蛋白转运蛋白识别并指导货物降解。在细菌感染过程中，内体蛋白转运蛋白转运蛋白的迁移导致宿主细胞不良环境中的细菌存活。 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来研究这种迁移的调节机制。该项目将包括通过新课程AccelateStem招募来自附近一所历史悠久的黑人学院，贝内特学院的高级本科生。该计划将在弗吉尼亚理工大学提供为期两年的培训承诺，以加速的M.S.结束。程度。 AccelateStem的目标是为学生提供所需的培训和资源，以增强他们在学术和非学术计划中的竞争力。该项目旨在确定有关调节内体货物转运蛋白TOM1的分子机制。 TOM1通过两个域（VHS和GAT）结合泛素化货物。泛素化的货物贩运可能需要一个高度组成的DXXLL分类图案，位于TOM1 VHS域的下游。有趣的是，含TOM1 DXXLL的区域已被证明是磷酸化的，这表明具有潜在的调节机制。 Shigella Flexneri细菌会产生5-磷酸磷脂酰肌醇（PTDINS5P），促进TOM1募集到内体。这导致内体成熟延迟，蛋白质更新减少以及宿主细胞内细菌存活的增强。通过等温滴定量热法，NMR光谱，分子动力学模拟和基于细胞的实验，Capelluto团队将测试含DXXLL的区域是否可以增强TOM1的货物运输功能以及其磷酸化是否起着调节作用。在病理情况下，如果局部酸化和PTDINS5P依赖性的膜结合影响Tom1在flexneri感染期间，团队将在分子和细胞水平上研究局部酸化和PTDINS5P依赖性膜结合。这些发现将在物理和病理背景下提高我们对细胞内蛋白质运输的理解。该奖项反映了NSF的法定任务，并通过使用基金会的知识分子优点和更广泛的影响评估标准来评估，被认为是珍贵的支持。