A Novel Domain Regulating Degradation of the Glomerular Slit Diaphragm Protein Podocin in Cell Culture Systems

Gödel, Markus; Ostendorf, Benjamin N.; Baumer, Jessica; Weber, Katrin; Huber, Tobias B.

doi:10.1371/journal.pone.0057078

Cited by 11 publications

(14 citation statements)

References 37 publications

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“…Then, by confocal microscopy, we confirmed the absence of Pod R138Q in the late endosome/lysosome compartment labelled with CD63 ( Figure 4B). Conversely, Pod wt is predominantly present in this compartment, as already described (13). Taken together, our data suggest that Pod wt is mainly degraded in lysosomes, in contrast to Pod R138Q , which is exclusively degraded by the proteasome.…”

Section: Resultssupporting

confidence: 87%

“…This same model may apply for misfolded Pod wt , since it also interacts with Cnx and its glycosylated forms are enriched after Kif treatment. Nevertheless, the increased evels of Pod wt following NH 4 Cl addition, together with the lack of response to Bz at short times, uggest that Pod wt is mainly degraded in the endosome/lysosome compartment, a finding which is in accordance with results from other authors (13).…”

Section: Discussionsupporting

confidence: 91%

“…However, an alternative transmembrane topology has been described for stomatin and podocin, particularly when a conserved proline residue, critical for the kink of the hairpin topology, found at position 118 in podocin, is mutated, and has been linked to N-glycosylated forms of these proteins (11,12). In addition, although the stability and degradation of podocin has been associated with a short internalization motif located in its C-terminus and to an interaction with the ubiquitin ligase Ubr4 (13,14), the mechanisms of Pod R138Q degradation are still unexplored. In this study, we report the degradation pathways followed by Pod R138Q , which might aid to establish new therapeutic strategies.…”

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confidence: 99%

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Endoplasmic reticulum–retained podocin mutants are massively degraded by the proteasome

Serrano-Pérez

Tilley

Névo

et al. 2018

Journal of Biological Chemistry

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Podocin is a key component of the slit diaphragm in the glomerular filtration barrier, and mutations in the podocin-encoding gene NPHS2 are a common cause of hereditary steroid-resistant nephrotic syndrome. A mutant allele encoding podocin with a p.R138Q amino acid substitution is the most frequent pathogenic variant in European and North American children, and the corresponding mutant protein is poorly expressed and retained in the endoplasmic reticulum (ER) both in vitro and in vivo. To better understand the defective trafficking and degradation of this mutant, we generated human podocyte cell lines stably expressing podocin wt or podocin R138Q . Although it has been proposed that podocin has a hairpin topology, we present evidence for podocin R138Q N-glycosylation, suggesting that most of the protein has a transmembrane topology. We find that N-glycosylated podocin R138Q has a longer half-life than non-glycosylated podocin R138Q , and that the latter is far more rapidly degraded than podocin wt . Consistent with its rapid degradation, podocin R138Q is exclusively degraded by the proteasome, whereas podocin wt is degraded by both the proteasomal and the lysosomal proteolytic machineries. In addition, we demonstrate an enhanced interaction of podocin R138Q with calnexin as the mechanism of ER retention. Calnexin knock-down enriches the podocin R138Q non-glycosylated fraction, whereas preventing exit from the calnexin cycle increases the glycosylated fraction. Altogether, we propose a model in which hairpin podocin R138Q is rapidly degraded by the proteasome, whereas transmembrane podocin R138Q degradation is delayed due to entry into the calnexin cycle.Nephrotic syndrome (NS) is clinically characterized by proteinuria, edema, hypoalbuminemia and hyperlipidemia, and is a consequence of glomerular filtration barrier (GFB) dysfunction. The prognosis of steroid-resistant nephrotic syndrome (SRNS) is poor, with a high proportion of patients rapidly developing end-stage renal disease, requiring dialysis or transplantation (1,2). The GFB is comprised principally of podocytes, specialised epithelial cells which interdigitate at junctions known as slit diaphragms (SDs). Mutations in the NPHS2 gene, encoding the slit diaphragm (SD) protein podocin, are the most frequent monogenic cause of SRNS in childhood (3,4 Podocin R138Q quality control and ERAD North America (4), and is associated with an earlyonset and rapidly progressing form of the disease (5). In accordance with the severe clinical phenotype, podocin p.R138Q (Pod R138Q ) is retained in the ER of podocytes and does not reach the SD, thus impairing correct functioning of the GFB (6,7).Podocin belongs to the stomatin and prohibitin homology domain (PHB) protein family and is specifically expressed in podocytes. It has been proposed that podocin acts as a molecular scaffold for other SD proteins in lipid raft membrane subdomains. For example, podocin interacts with both nephrin and CD2AP through its carboxy (C)-terminus, and participates in various signaling...

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Section: Resultssupporting

confidence: 87%

Section: Discussionsupporting

confidence: 91%

mentioning

confidence: 99%

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Endoplasmic reticulum–retained podocin mutants are massively degraded by the proteasome

Serrano-Pérez

Tilley

Névo

et al. 2018

Journal of Biological Chemistry

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“…Disease-causing mutations in the NPHS2 gene in patients with SRNS were shown to not only cause misfolding of podocin and alter its localization, but they also altered the trafficking of normal nephrin to the plasma membrane [23,24]. Moreover, several domains of podocin have been demonstrated to facilitate specific functions of this protein, such as oligomerization in lipid raft microdomains, membrane association and interaction with other SD components, including nephrin and CD2AP [24,25]. …”

Section: Introductionmentioning

confidence: 99%

Co-Inheritance of Functional Podocin Variants with Heterozygous Collagen IV Mutations Predisposes to Renal Failure

Stefanou

Pieri

Savva

et al. 2015

Nephron

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Background/Aims: A subset of patients who present with proteinuria and are diagnosed with focal segmental glomerulosclerosis (FSGS) have inherited heterozygous COL4A3/A4 mutations and are also diagnosed with thin basement membrane nephropathy (TBMN-OMIM: 141200). Two studies showed that co-inheritance of NPHS2-p.Arg229Gln, a podocin variant, may increase the risk for proteinuria and renal function decline. Methods: We hypothesized that additional podocin variants may exert a similar effect. We studied genetically a well-characterized Cypriot TBMN patient cohort by re-sequencing the NPHS2 coding region. We also performed functional studies in cell culture experiments, investigating the interaction of podocin variants with itself and with nephrin. Results: Potentially disease-modifying podocin variants were searched for by analyzing NPHS2 in 35 ‘severe' TBMN patients. One non-synonymous variant, p.Glu237Gln, was detected. Both variants, p.Arg229Gln and p.Glu237Gln, were tested in a larger cohort of 122 TBMN patients, who were categorized as ‘mild' or ‘severe' based on the presence of microscopic hematuria alone or combined with chronic renal failure and/or proteinuria. Seven ‘severe' patients carried either of the 2 variants; none was present in the ‘mild' patients (p = 0.05, Pearson χ²). The 7 carriers belong in 2 families segregating mutation COL4A3-p.Gly1334Glu. Inheritance of the wild-type (WT) and mutant alleles correlated with the phenotype (combined concordance probability 0.003). Immunofluorescence (IF) experiments after dual co-transfection of WT and mutant podocin suggested altered co-localization of mutant homodimers. IF experiments after co-transfection of WT podocin and nephrin showed normal membrane localization, while both podocin variants interfered with normal trafficking, demonstrating perinuclear staining. Immunoprecipitation experiments showed stronger binding of mutant podocin to WT podocin or nephrin. Conclusion: The results support the hypothesis that certain hypomorphic podocin variants may act as adverse genetic modifiers when co-inherited with COL4A3/A4 mutations, thus predisposing to FSGS and severe kidney function decline.

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“…In contrast, however, Godel et al . demonstrated that a fraction of podocin resides in the CD63/LAMP3-positive late endosomal compartment and has limited co-localized with EEA1 in transiently co-transfected HeLa cells33. We recently reported considerable co-localization of both podocin and nephrin with Rab7 and LAMP1 in podocyte-specific cathepsin D knock-out mice (CD pdKO ), compared with control mice34, suggesting that podocin and nephrin are mainly localized in the late endosomes and lysosomes of CD pdKO mouse podocyte cell bodies.…”

Section: Discussionmentioning

confidence: 99%

Sorting Nexin 9 facilitates podocin endocytosis in the injured podocyte

Sasaki

Hidaka

Ueno

et al. 2017

Sci Rep

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The irreversibility of glomerulosclerotic changes depends on the degree of podocyte injury. We have previously demonstrated the endocytic translocation of podocin to the subcellular area in severely injured podocytes and found that this process is the primary disease trigger. Here we identified the protein sorting nexin 9 (SNX9) as a novel facilitator of podocin endocytosis in a yeast two-hybrid analysis. SNX9 is involved in clathrin-mediated endocytosis, actin rearrangement and vesicle transport regulation. Our results revealed and confirmed that SNX9 interacts with podocin exclusively through the Bin–Amphiphysin–Rvs (BAR) domain of SNX9. Immunofluorescence staining revealed the expression of SNX9 in response to podocyte adriamycin-induced injury both in vitro and in vivo. Finally, an analysis of human glomerular disease biopsy samples demonstrated strong SNX9 expression and co-localization with podocin in samples representative of severe podocyte injury, such as IgA nephropathy with poor prognosis, membranous nephropathy and focal segmental glomerulosclerosis. In conclusion, we identified SNX9 as a facilitator of podocin endocytosis in severe podocyte injury and demonstrated the expression of SNX9 in the podocytes of both nephropathy model mice and human patients with irreversible glomerular disease.

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A Novel Domain Regulating Degradation of the Glomerular Slit Diaphragm Protein Podocin in Cell Culture Systems

Cited by 11 publications

References 37 publications

Endoplasmic reticulum–retained podocin mutants are massively degraded by the proteasome

Endoplasmic reticulum–retained podocin mutants are massively degraded by the proteasome

Co-Inheritance of Functional Podocin Variants with Heterozygous Collagen IV Mutations Predisposes to Renal Failure

Sorting Nexin 9 facilitates podocin endocytosis in the injured podocyte

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