Investigating the Biogenesis and Degradation of Polycystin 2 Missense Mutants

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent inherited disorder and cause of end-stage renal disease. ADPKD arises from mutations in PKD1 and PKD2, which encode polycystin 1 (PC1) and polycystin 2 (PC2). These mutations lead to aberrant signaling, cell proliferation, and fluid secretion. Given its size, complexity, and primary localization to the endoplasmic reticulum (ER), we hypothesize that PC2 missense mutants may misfold and be degraded by the ER-associated degradation (ERAD) pathway. Methods: Disease-causing PC2 variants that were computationally predicted to be folding defective were expressed in S. cerevisiae and HEK293 cells to evaluate their stability and function. Stability and poly-ubiquitination levels were measured in the presence or absence of a proteasome inhibitor (MG132). TEVC studies in Xenopus oocytes and cell surface biotinylation revealed PC2 activity and cell surface residence. We tested the ability of PC2 missense variants to rescue growth in yeast lacking two plasma membrane potassium transporters and determined response to cold temperature correction and chemical chaperones. Results: Select PC2 missense mutants exhibited more poly-ubiquitination and were degraded faster than wild-type PC2 in both yeast and HEK293 cells. Treatment with MG132 increased poly-ubiquitinated protein levels, suggesting proteasomal turnover of these mutants. The yeast growth assay reports on protein folding, assembly, trafficking, and activity; some mutants failed to rescue yeast growth, while others exhibited normal growth. Incubation at lower temperatures improved growth of select mutants, perhaps indicating a potential for pharmacological rescue. Some yeast growth defects were recapitulated in oocytes, validating our yeast model system. Finally, surface biotinylation of PC2 variants in HEK293 cells showed reduced amounts of unstable PC2 missense variants at the cell surface, which may contribute to the ADPKD phenotype. Conclusion: While some PC2 alleles are stable yet dysfunctional, suggesting trafficking defects or impaired ion transport as the source of dysfunction, we find that select PC2 missense mutants are targeted for the ERAD pathway. Protein folding modulators are a rapidly expanding therapeutic strategy for ERAD-related diseases, and based on our data some PC2 mutants would be amenable to pharmacological correction. Funding: NIDDK Support - NIDDK Support, NIDDK Support