Sorin V. Fedeles

A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation

Autosomal dominant polycystic liver disease results from mutations in PRKCSH or SEC63. The respective gene products, glucosidase IIβ and SEC63p, function in protein translocation and quality control pathways in the endoplasmic reticulum. Here we show that glucosidase IIβ and Sec63p are required in mice for adequate expression of a functional complex of the polycystic kidney disease gene products, polycystin-1 and polycystin-2. We find that polycystin-1 is the rate-limiting component of this complex and that there is a dose-response relationship between cystic dilation and levels of functional polycystin-1 following mutation of Prkcsh or Sec63. Reduced expression of polycystin-1 also serves to sensitize the kidney to cyst formation resulting from mutations in Pkhd1, the recessive polycystic kidney disease gene. Finally, we show that proteasome inhibition increases steady-state levels of polycystin-1 in cells lacking glucosidase IIβ and that treatment with a proteasome inhibitor reduces cystic disease in orthologous gene models of human autosomal dominant polycystic liver disease.

Different effects of Sec61α, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells

Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.

Altered trafficking and stability of polycystins underlie polycystic kidney disease

The most severe form of autosomal dominant polycystic kidney disease occurs in patients with mutations in the gene (PKD1) encoding polycystin-1 (PC1). PC1 is a complex polytopic membrane protein expressed in cilia that undergoes autoproteolytic cleavage at a G protein-coupled receptor proteolytic site (GPS). A quarter of PKD1 mutations are missense variants, though it is not clear how these mutations promote disease. Here, we established a cell-based system to evaluate these mutations and determined that GPS cleavage is required for PC1 trafficking to cilia. A common feature among a subset of pathogenic missense mutations is a resulting failure of PC1 to traffic to cilia regardless of GPS cleavage. The application of our system also identified a missense mutation in the gene encoding polycystin-2 (PC2) that prevented this protein from properly trafficking to cilia. Using a Pkd1-BAC recombineering approach, we developed murine models to study the effects of these mutations and confirmed that only the cleaved form of PC1 exits the ER and can rescue the embryonically lethal Pkd1-null mutation. Additionally, steady-state expression levels of the intramembranous COOH-terminal fragment of cleaved PC1 required an intact interaction with PC2. The results of this study demonstrate that PC1 trafficking and expression require GPS cleavage and PC2 interaction, respectively, and provide a framework for functional assays to categorize the effects of missense mutations in polycystins.

Isolated polycystic liver disease genes define effectors of polycystin-1 function

Dominantly inherited isolated polycystic liver disease (PCLD) consists of liver cysts that are radiologically and pathologically identical to those seen in autosomal dominant polycystic kidney disease, but without clinically relevant kidney cysts. The causative genes are known for fewer than 40% of PCLD index cases. Here, we have used whole exome sequencing in a discovery cohort of 102 unrelated patients who were excluded for mutations in the 2 most common PCLD genes, PRKCSH and SEC63, to identify heterozygous loss-of-function mutations in 3 additional genes, ALG8, GANAB, and SEC61B. Similarly to PRKCSH and SEC63, these genes encode proteins that are integral to the protein biogenesis pathway in the endoplasmic reticulum. We inactivated these candidate genes in cell line models to show that loss of function of each results in defective maturation and trafficking of polycystin-1, the central determinant of cyst pathogenesis. Despite acting in a common pathway, each PCLD gene product demonstrated distinct effects on polycystin-1 biogenesis. We also found enrichment on a genome-wide basis of heterozygous mutations in the autosomal recessive polycystic kidney disease gene PKHD1, indicating that adult PKHD1 carriers can present with clinical PCLD. These findings define genetic and biochemical modulators of polycystin-1 function and provide a more complete definition of the spectrum of dominant human polycystic diseases.

N-Glycosylation Determines the Abundance of the Transient Receptor Potential Channel TRPP2

Background: Transient receptor potential channel TRPP2 is a glycoprotein mutated in polycystic kidney disease. Results: Five N-glycosylation sites in TRPP2 and glucosidase II-mediated glycan trimming determine TRPP2 protein abundance. Conclusion: N-Glycosylation of TRPP2 in the first extracellular loop is required for efficient protein translation and stability. Significance: The mechanisms controlling the biogenesis of TRPP2 may provide insights into the pathogenesis of polycystic kidney and polycystic liver disease. Glycosylation plays a critical role in the biogenesis and function of membrane proteins. Transient receptor potential channel TRPP2 is a nonselective cation channel that is mutated in autosomal dominant polycystic kidney disease. TRPP2 has been shown to be heavily N-glycosylated, but the glycosylation sites and the biological role of N-linked glycosylation have not been investigated. Here we show, using a combination of mass spectrometry and biochemical approaches, that native TRPP2 is glycosylated at five asparagines in the first extracellular loop. Glycosylation is required for the efficient biogenesis of TRPP2 because mutations of the glycosylated asparagines result in strongly decreased protein expression of the ion channel. Wild-type and N-glycosylation-deficient TRPP2 is degraded in lysosomes, as shown by increased TRPP2 protein levels upon chemical inhibition of lysosomal degradation. In addition, using pharmacological and genetic approaches, we demonstrate that glucosidase II (GII) mediates glycan trimming of TRPP2. The non-catalytic β subunit of glucosidase II (GIIβ) is encoded by PRKCSH, one of the genes causing autosomal dominant polycystic liver disease (ADPLD). The impaired GIIβ-dependent glucose trimming of TRPP2 glycosylation in ADPLD may explain the decreased TRPP2 protein expression in Prkcsh−/− mice and the genetic interaction observed between TRPP2 and PRKCSH in ADPLD. These results highlight the biological importance of N-linked glycosylation and GII-mediated glycan trimming in the control of biogenesis and stability of TRPP2.

Sec63 and Xbp1 regulate IRE1α activity and polycystic disease severity

The HSP40 cochaperone SEC63 is associated with the SEC61 translocon complex in the ER. Mutations in the gene encoding SEC63 cause polycystic liver disease in humans; however, it is not clear how altered SEC63 influences disease manifestations. In mice, loss of SEC63 induces cyst formation both in liver and kidney as the result of reduced polycystin-1 (PC1). Here we report that inactivation of SEC63 induces an unfolded protein response (UPR) pathway that is protective against cyst formation. Specifically, using murine genetic models, we determined that SEC63 deficiency selectively activates the IRE1α-XBP1 branch of UPR and that SEC63 exists in a complex with PC1. Concomitant inactivation of both SEC63 and XBP1 exacerbated the polycystic kidney phenotype in mice by markedly suppressing cleavage at the G protein-coupled receptor proteolysis site (GPS) in PC1. Enforced expression of spliced XBP1 (XBP1s) enhanced GPS cleavage of PC1 in SEC63-deficient cells, and XBP1 overexpression in vivo ameliorated cystic disease in a murine model with reduced PC1 function that is unrelated to SEC63 inactivation. Collectively, the findings show that SEC63 function regulates IRE1α/XBP1 activation, SEC63 and XBP1 are required for GPS cleavage and maturation of PC1, and activation of XBP1 can protect against polycystic disease in the setting of impaired biogenesis of PC1.

Steviol retards renal cyst growth through reduction of CFTR expression and inhibition of epithelial cell proliferation in a mouse model of polycystic kidney disease.

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) is associated with cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel leading to renal failure for which no effective treatment is currently available. We previously reported that steviol retards Madin-Darby canine kidney (MDCK) cyst enlargement by inhibiting CFTR channel activity and promoting proteasomal-mediated CFTR degradation. It is imperative to examine the effect of steviol in animal models of ADPKD. Therefore, we examined the effect of steviol on renal cyst growth in an orthologous mouse model of human ADPKD (Pkd1(flox/flox):Pkhd1-Cre). The results showed that daily treatment with both 200mg/kg BW of steviol and 1000mg/kg BW of stevioside for 14 days markedly decreased kidney weight and cystic index in these mice. However, only steviol markedly reduced blood urea nitrogen and creatinine values. Steviol also reduced cell proliferation but had no effect on cell apoptosis. In addition, steviol suppressed CFTR and mTOR/S6K expression in renal cyst-lining epithelial cells. Interestingly, steviol was found to stimulate AMP-activated protein kinase (AMPK). Our findings indicate that steviol slows cyst progression in ADPKD mouse model, in part, through the activation of AMPK which subsequently inhibits CFTR chloride channel expression and inhibits renal epithelial cell proliferation via mTOR/S6K pathway. Most importantly, steviol could markedly improve kidney function in a mouse model of ADPKD. Steviol thus has potential application for further development as a therapeutic compound for the treatment of polycystic kidney disease.

Cell polarity and cystic kidney disease

Epithelial cell polarity is essential for organ development; aberrations in this process have been implicated in various diseases, including polycystic kidney disease. Establishment and maintenance of cell polarity is governed by a number of molecular processes and how these processes operate remains an interesting question. Conserved protein complexes guide both apical–basolateral polarity and planar cell polarity. In this review we discuss the recent findings that provide insights into polarity mechanisms and the intriguing crosstalk between apical–basolateral polarity and planar cell polarity, and their relationship to cystic kidney disease.

Essential Role of X-Box Binding Protein-1 during Endoplasmic Reticulum Stress in Podocytes

Podocytes are terminally differentiated epithelial cells that reside along the glomerular filtration barrier. Evidence suggests that after podocyte injury, endoplasmic reticulum stress response is activated, but the molecular mechanisms involved are incompletely defined. In a mouse model, we confirmed that podocyte injury induces endoplasmic reticulum stress response and upregulated unfolded protein response pathways, which have been shown to mitigate damage by preventing the accumulation of misfolded proteins in the endoplasmic reticulum. Furthermore, simultaneous podocyte-specific genetic inactivation of X-box binding protein-1 (Xbp1), a transcription factor activated during endoplasmic reticulum stress and critically involved in the untranslated protein response, and Sec63, a heat shock protein-40 chaperone required for protein folding in the endoplasmic reticulum, resulted in progressive albuminuria, foot process effacement, and histology consistent with ESRD. Finally, loss of both Sec63 and Xbp1 induced apoptosis in podocytes, which associated with activation of the JNK pathway. Collectively, our results indicate that an intact Xbp1 pathway operating to mitigate stress in the endoplasmic reticulum is essential for the maintenance of a normal glomerular filtration barrier.

Is it Time to Fold the Cysts Away

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2, encoding polycystin-1 and polycystin-2, respectively. Optimizing the folding environment for polycystin-1 missense mutations may have a critical effect on the progression of ADPKD in animal models and could potentially lead to tangible therapeutic options for subgroups of ADPKD patients.