Revealing the regulatory mechanisms of endosomal cargo transporters

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.