Revealing the regulatory mechanisms of endosomal cargo transporters

本计画将探讨内体蛋白质转运蛋白在生理与病理上的调控。 对这些转运蛋白的调控机制知之甚少，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。这些修饰的受体，称为货物，被递送到内体，其中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重新定位的调控机制。该项目将包括通过一个新的项目“加速干”，从附近一所历史悠久的黑人大学班尼特学院招募高年级本科生。 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MS结束。℃下AccelerateSTEM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。 该项目旨在确定内体货物转运蛋白TOM 1调控的分子机制。TOM 1通过两个结构域VHS和GAT结合泛素化货物。位于TOM 1 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。有趣的是，TOM 1 DXXLL包含区域已被证明是磷酸化的，这表明了一种潜在的调控机制。细菌福氏志贺菌产生磷脂酰肌醇5-磷酸（PtdIns 5 P），促进TOM 1募集到内体。这导致内体成熟延迟、蛋白质周转减少和宿主细胞内细菌存活增强。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的组合，Capelluto团队将测试含有DXXLL的区域是否增强TOM 1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，研究小组将在分子和细胞水平上研究局部酸化和PtdIns 5 P依赖性膜结合是否会影响TOM 1在S.弗氏菌感染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NSF的法定使命，并被认为值得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来支持。

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.