My long-term goal has been to investigate the quality control mechanisms that regulate the levels of disease- causing proteins in the kidney. My most recent project has focused on polycystin 2 (PC2). PC2 plays a vital role in regulating calcium homeostasis, but mutations in the PKD2 gene, which encodes PC2, can lead to autosomal dominant polycystic kidney disease (ADPKD). ADPKD is characterized by the formation of large fluid-filled cysts in the kidney, thereby decreasing kidney function. There is no cure for this disease. However, a deeper understanding of how mutations alter the fate of the PC2 protein will provide insight into novel treatments for ADPKD. Interestingly, the majority of PC2 is found in the endoplasmic reticulum (ER), but some of the protein travels through the secretory pathway to the plasma membrane. One pathway that controls the fate of proteins in the ER is endoplasmic reticulum-associated degradation (ERAD). During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated from the ER membrane for degradation by the cytoplasmic proteasome. The importance of ERAD to human health is highlighted by the discovery of >70 disease-associated proteins that are targeted to this pathway, many of which are channels and transporters. Indeed, my preliminary data suggest?for the first time?that ERAD plays a central role in regulating the biogenesis of select PC2 missense mutants that are disease-causing. To elucidate how these proteins are targeted for destruction, I will use a powerful combination of genetic, biochemical, and physiological methods and will develop new experimental tools. My overall hypothesis is that select missense mutations in PC2 are targeted to the ERAD pathway by molecular chaperones, for which therapeutics are currently being developed, as well as by other components of the ?protein quality control? machinery in the cell. To test this hypothesis, the specific aims of this proposal are: (1) To establish a yeast PC2 expression system, which allow me to coopt facile genetic approaches and then define how PC2 missense mutants are targeted for ERAD. Discoveries from this attack will next be confirmed in renal epithelial cell culture systems; and (2) To develop a new yeast screen in which novel genetic modifiers of PC2 protein turnover can be identified and in which a whole genomic analysis can be undertaken. Hits from this screen will be evaluated in the future in cell culture and rodent models. This project will identify the molecular mechanisms that lead to ADPKD in patients who carry a defined group of PC2 mutations and, in the long-term, uncover a range of potential therapeutics.
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders and is caused by mutations in the genes encoding polycystin 1 (PC1) and 2 (PC2). Understanding the nature of the defects resulting from different mutations in these proteins is critical for the development of therapies to treat ADPKD. Based on new preliminary data, the experiments outlined in this proposal will identify how select polycystin 2 disease-causing mutants are targeted for degradation with the long-term goal of uncovering drug targets to treat ADPKD.
