Revealing the regulatory mechanisms of endosomal cargo transporters

这个项目将研究生理和病理情况下内体蛋白转运蛋白的调节。对这些转运蛋白的调控机制知之甚少，这在该领域构成了一个重大的瓶颈。细胞修饰它们的膜蛋白受体，作为内化的信号。这些修饰的受体被称为货物，被运送到内体，在那里内体蛋白转运体识别和引导货物降解。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生存。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次搬迁的调控机制。该项目将包括通过一个名为AccelerateSTEM的新项目，从附近一所历史上一直是黑人的学院班尼特学院(Bennett College)招收高年级本科生。该项目将在弗吉尼亚理工大学提供为期两年的培训承诺，最终获得更快的理科硕士学位。AccelerateSTEM的目标是为学生提供必要的培训和资源，以增强他们在学术和非学术项目中的竞争力。该项目旨在确定调控内体货物转运蛋白TOM1的分子机制。TOM1通过VHS和GAT两个结构域与泛素化的货物结合。泛素化的货物贩运可能需要一个高度保守的DXXLL分选基序，位于TOM1 VHS结构域的下游。有趣的是，含有TOM1 DXXLL的区域已被证明是磷酸化的，这表明了一种潜在的调控机制。福氏志贺菌产生磷脂酰肌醇5-磷酸(PtdIns5P)，促进TOM1募集到内体。这会导致内体成熟延迟，蛋白质周转减少，并提高宿主细胞内细菌的存活率。通过结合等温滴定热法、核磁共振光谱、分子动力学模拟和基于细胞的实验，Capelluto团队将测试DXXLL区域是否增强TOM1的货物运输功能，以及它的磷酸化是否起到调制作用。在病理场景中，该团队将在分子和细胞水平上研究局部酸化和依赖PtdIns5P的膜结合是否影响TOM1的S在福氏志贺菌感染期间的货物运输功能。这些发现将促进我们对生理和病理情况下细胞内蛋白质运输的理解。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.