Identification of Polycystin 2 Missense Mutants Targeted for Endoplasmic Reticulum-Associated Degradation

Academic Background

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disorder that ultimately leads to end-stage renal disease. ADPKD primarily arises from mutations in the PKD1 and PKD2 genes, which encode Polycystin 1 (PC1) and Polycystin 2 (PC2), respectively. PC2 is a nonselective cation channel, and disease-linked mutations disrupt its normal functions, including signaling and fluid secretion. Although PC1 and PC2 are known as causative factors of ADPKD, the mechanisms by which most disease-associated PC2 missense mutations lead to ADPKD remain unclear. In particular, whether PC2 missense mutations impair its folding and lead to degradation via the Endoplasmic Reticulum-Associated Degradation (ERAD) pathway has not been fully investigated.

This study aims to explore whether disease-related PC2 missense mutations affect its folding and result in degradation through the ERAD pathway. To achieve this, researchers developed a new yeast PC2 expression system and used it to study the biogenesis process of PC2. They also examined the stability, ubiquitination levels, and cell surface localization of two disease-associated PC2 mutants (D511V and R322Q) in yeast and HEK293 cells, and explored whether the folding of these mutants could be corrected under low-temperature conditions.

Paper Source

This paper was co-authored by Christopher J. Guerriero, Marcelo D. Carattino, Katherine G. Sharp, Luke J. Kantz, Nikolay P. Gresko, Michael J. Caplan, and Jeffrey L. Brodsky. The authors are affiliated with the Department of Biological Sciences at the University of Pittsburgh, the Departments of Medicine and Cell Biology at the University of Pittsburgh, and the Department of Cellular and Molecular Physiology at Yale University. The paper was first published on December 23, 2024, in the journal American Journal of Physiology-Cell Physiology, with the DOI: 10.1152/ajpcell.00776.2024.

Research Process and Results

1. Development of the Yeast PC2 Expression System

The researchers first developed a new yeast PC2 expression system to study the biogenesis process of PC2. They expressed human PC2 with an N-terminal triple HA tag in yeast and constructed both low-copy (CEN) and high-copy (2μ) expression vectors. By comparing the effects of different copy numbers on yeast growth, they found that high-copy vectors slowed yeast growth, so subsequent experiments mainly used low-copy vectors.

To verify the expression and localization of PC2 in yeast, the researchers performed glycosylation analysis on PC2. The results showed that PC2 was glycosylated in both yeast and HEK293 cells, and treatment with endoglycosidase H (Endo H) removed the glycosylated forms of PC2, indicating that PC2 correctly localized to the endoplasmic reticulum (ER) and inserted into the ER membrane. Further live-cell microscopy confirmed that the PC2-GFP fusion protein was primarily localized in the ER in both yeast and HEK293 cells.

2. Stability and ERAD Targeting of PC2 Mutants

The researchers first compared the steady-state expression levels of wild-type PC2 and two disease-associated mutants (D511V and R322Q) in yeast. The results showed that the expression level of the D511V mutant was significantly reduced, while the expression level of the R322Q mutant was similar to that of wild-type PC2. Further cycloheximide chase experiments indicated that the D511V mutant was unstable in yeast, whereas the stability of the R322Q mutant was similar to that of wild-type PC2.

To investigate whether the D511V mutant was degraded via the ERAD pathway, the researchers treated the cells with the proteasome inhibitor MG132. The results showed that MG132 treatment significantly stabilized the D511V mutant, indicating that it was indeed degraded via the ERAD pathway. Further ubiquitination experiments showed that the ubiquitination level of the D511V mutant in yeast was significantly higher than that of wild-type PC2 and the R322Q mutant.

3. Functional Activity Assay of PC2 in Yeast

To test whether PC2 forms functional channels in yeast, the researchers used a quantitative growth assay based on yeast. They employed a yeast strain lacking endogenous potassium transporters (Trk1 and Trk2) and tested the function of PC2 in low-potassium medium. The results showed that wild-type PC2 could not support yeast growth in low-potassium medium, while a gain-of-function mutant of PC2 (PC2_2a) could support yeast growth. However, neither the D511V nor the R322Q mutant in the PC2_2a background could support yeast growth, indicating that these mutants had lost channel function.

4. Study of PC2 Mutants in HEK293 Cells

To verify whether the results in yeast were applicable to higher eukaryotic cells, the researchers expressed PC2-GFP and its mutants in HEK293 cells. The results showed that the steady-state expression levels of both the D511V and R322Q mutants were significantly reduced in HEK293 cells. Further cycloheximide chase experiments indicated that both mutants were unstable in HEK293 cells, and their ubiquitination levels were significantly higher than those of wild-type PC2.

The researchers also detected the localization of PC2 mutants on the surface of HEK293 cells using cell surface biotinylation assays. The results showed that the presence of D511V and R322Q mutants on the cell surface was significantly reduced, indicating that the folding defects of these mutants affected their transport to the cell surface. However, under low-temperature (26°C) conditions, the total expression levels and cell surface localization of both mutants were partially restored, suggesting that their folding defects could be corrected by low temperatures.

5. Functional Testing of PC2 in Xenopus Oocytes

To further study the function of PC2 mutants, the researchers expressed a gain-of-function mutant of PC2 (F604P) and its disease-associated mutants (D511V and R322Q) in Xenopus oocytes and measured their currents using Two-Electrode Voltage Clamp (TEVC) experiments. The results showed that the D511V mutant completely lost channel function in oocytes, while the current of the R322Q mutant was significantly lower than that of the F604P mutant but still higher than that of the uninjected control group. This result suggests that the folding defect of the R322Q mutant may be partially corrected under low-temperature conditions, thereby restoring partial channel function.

Conclusions and Implications

This study shows that certain PC2 missense mutations cause protein folding defects and are degraded via the ERAD pathway. The researchers developed a new yeast PC2 expression system to study the biogenesis and function of PC2 and found that the D511V mutant is degraded via the ERAD pathway in both yeast and HEK293 cells. Additionally, the R322Q mutant exhibited characteristics of ERAD targeting in HEK293 cells but was relatively stable in yeast. Under low-temperature conditions, the folding and cell surface localization of both mutants were partially restored, suggesting that their folding defects can be corrected through pharmacological intervention.

The highlight of this study is the development of a new yeast model system for rapidly screening and identifying loss-of-function mutants of PC2 and revealing the mechanism by which certain PC2 missense mutations are degraded via the ERAD pathway. Moreover, the study indicates that low temperatures can partially correct the folding defects of PC2 mutants, providing new ideas for developing therapeutic strategies for ADPKD.

Research Value

This study not only reveals the mechanism by which PC2 missense mutations are degraded via the ERAD pathway but also provides potential new strategies for the treatment of ADPKD. By developing a yeast model system, researchers can rapidly screen and identify loss-of-function mutants of PC2, providing an important experimental platform for future drug development. Additionally, the study shows that low temperatures can partially correct the folding defects of PC2 mutants, offering a theoretical basis for developing protein folding correctors for ADPKD.

This research provides new insights into understanding the pathogenic mechanisms of PC2 missense mutations and opens up new directions for developing therapeutic strategies for ADPKD.