Defective Trafficking of Nephrin Missense Mutants Rescued by a Chemical Chaperone
Xiao Li Liu*,
Stefania Cotta Doné*,
Kunimasa Yan,
Pekka Kilpeläinen,
Timo Pikkarainen* and
Karl Tryggvason*
*Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden; Department of Pediatrics, Kyorin University School of Medicine, Mitaka, Tokyo, Japan; and Biocenter Oulu, University of Oulu, Oulu, Finland
Correspondence to Dr. Karl Tryggvason, Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden. Phone: 46-8-524-877-20; Fax: 46-8-313-445; E-mail: karl.tryggvason{at}mbb.ki.se
ABSTRACT. The nephrin gene (NPHS1) is mutated in congenitalnephrotic syndrome of the Finnish type. Most mutations foundin non-Finnish patients are missense mutations. The most commonconsequence of missense mutations in congenital nephrotic syndromeis a defect in intracellular transport and retention of themutant proteins in the endoplasmic reticulum (ER), possiblyas a result of misfolding and unfavored conformation. Becausesodium 4-phenylbutyrate has been shown to function as a chemicalchaperone and to correct the cellular trafficking of severalmislocalized or misfolded mutant plasma membrane proteins, theeffects of this compound on the missense mutants identifiedin patients with congenital nephrotic syndrome of the Finnishtype were investigated. This study was performed using humanembryonic kidney 293 cells stably expressing wild-type or missensenephrin mutants trapped in the ER. Immunofluorescence microscopyand cell surface biotinylation showed that treatment with sodium4-phenylbutyrate rescued several of the missense mutants fromthe ER to the cell surface. All of the rescued mutants werefound to be able to interact with Neph1. Furthermore, theirtyrosine phosphorylation was rapidly induced by clustering withanti-nephrin antibodies, suggesting that the rescued mutantsmay be functionally intact.
Congenital nephrotic syndrome of the Finnish type (CNF) is anautosomal recessive disorder characterized by massive proteinuriaalready in utero and development of nephrosis soon after birth(1). Renal transplantation after intensive treatment, includingnephrectomy, peritoneal dialysis, and intravenous nutrition,is the only curative therapy for CNF (2). The causative genefor CNF has been cloned and shown to encode a novel podocyte-specifictype 1 membrane protein, nephrin, which belongs to the largeIg-like superfamily (3). Nephrin has an extracellular part consistingof eight Ig motifs followed by a fibronectin type III (FNIII)domain, a short transmembrane region, and a cytoplasmic C-terminalpart. The specific location of nephrin in the slit diaphragmsuggests that nephrin is the principal component of the slitdiaphragm structure (4) and that the renal failure in CNF isdirectly caused by the absence or abnormal function of nephrin.
More than 60 different mutations, including deletions; insertions;and nonsense, missense, splice site, and promoter mutations,have been identified in Finnish and non-Finnish patients withCNF. The most common mutations are missense mutations resultingin single amino acid substitutions. To date, 31 different missensemutations have been reported in CNF patients (5–7). Ourprevious study showed that the majority of the missense mutationslead to protein misfolding and consequently to the retentionof the mutants in the endoplasmic reticulum (ER). These mutantsare absent from the plasma membrane, which provides the molecularpathomechanism of the nephrotic syndrome in NPHS1 patients whocarry such missense mutations (8).
Sodium 4-phenylbutyrate (4-PBA) has been shown to function asa chemical chaperone that can correct the cellular traffickingof several mislocalized or misfolded mutant plasma membraneproteins, such as cystic fibrosis transmembrane conductanceregulator (CFTR) and 1-antitrypsin (9,10). In this study, weexamined the potential effect of 4-PBA on the trafficking ofnephrin missense mutants by using immunofluorescence microscopy,cell surface biotinylation, and Western blotting. It was demonstratedthat 4-PBA is able to rescue a number of the nephrin missensemutants to escape the ER quality control. The proteins are transportedto the plasma membrane, where they function indistinguishablyfrom wild-type nephrin, as judged by their ability to interactwith Neph1 and to become heavily tyrosine phosphorylated whenclustered by anti-nephrin antibodies.
Cell Culture and Generation of Stable Cell Lines
All cell culture media and supplements were purchased from Invitrogen(San Diego, CA). The human embryonic kidney cell line 293 (HEK-293;Qbiogene) was cultured in DMEM supplemented with 10% FBS, 100units/ml penicillin, and 100 mg/ml streptomycin. The stablecell line expressing wild-type nephrin has been described previously(8). Constructs encoding the missense mutants, as well as celllines stably expressing these proteins, were generated as describedpreviously (8). For maintaining nephrin expression, the cellswere grown in the presence of G418 (1 mg/ml) at 37°C.
Immunofluorescence Staining
All of the steps described below were performed at room temperature,unless indicated otherwise. Confluent cultures of the stablenephrin transfectants were incubated at 37°C in the absenceor presence of 10 mM 4-PBA (Fyrklövern Scandinavia AB,Mönsterås, Sweden) for 24 h. Subsequently, the cellswere trypsinized and cultured on glass coverslips overnightwith or without 4-PBA. The cells were then washed with PBS andfixed in 2% formaldehyde and 0.1% glutaraldehyde in PBS for15 min at 37°C. After fixation, the coverslips were washedtwice with PBS, blocked with 2% BSA and 1% casein hydroxylatein PBS buffer for 1 h, and then incubated for 1 h with the monoclonalanti-nephrin antibody 50A9 (11,12) diluted to 1:100 in the blockingsolution. After three washes, the cells were incubated withFITC-conjugated goat anti-mouse IgG (Dako, Glostrup, Denmark)for 1 h. Filamentous actin was stained with rhodamine-phalloidin(Molecular Probes, Leiden, the Netherlands). The coverslipswere analyzed using a DMRB Leica microscope and photographedwith a digital camera (Hamamatsu C4742-95).
Cell Surface Biotinylation and Western Blot Analyses
Confluent cultures of the different stable nephrin transfectantswere incubated for 24 h in the absence or presence of 4-PBA,as described above. Subsequently, the cells were plated onto60-mm culture dishes and cultured overnight. The following stepswere performed at 4°C, unless stated otherwise. After washingtwice with PBS Ca2+/Mg2+ (PBS containing 1 mM CaCl2 and 0.5mM MgCl2), the cells were incubated in 2 mM sulfo-NHS-SS-biotinin PBS Ca2+/Mg2+ for 2 h. The cells were washed three timeswith PBS Ca2+/Mg2+ containing 50 mM glycine to quench the unreactedbiotin and once with PBS Ca2+/Mg2+ to remove the excess of glycine.The cells were lysed in ice-cold RIPA buffer (150 mM NaCl, 50mM Tris-HCl, 1% Nonidet P-40, 1% sodium deoxycholate [pH 8.0])containing protease inhibitors (1 mM PMSF and 5 µg/mleach of aprotinin, antipain, pepstatin A, and leupeptin). Afteradjusting the volumes of the lysates to 1 ml with the RIPA buffer,biotinylated proteins were captured with streptavidin-agarosebeads (Immunopure, immobilized streptavidin; Pierce, Rockford,IL) by incubating overnight at 4°C with end-to-end mixing.The beads were then washed once with washing solution A (150mM NaCl, 50 mM Tris-HCl [pH 7.4], and 5 mM EDTA), twice withwashing solution B (500 mM NaCl, 50 mM Tris-HCl [pH 7.4], and5 mM EDTA), three times with washing solution C (500 mM NaCl,20 mM Tris-HCl [pH 7.4], and 0.2% BSA), and once with washingsolution D (10 mM Tris-HCl [pH 7.4]). The biotinylated proteinswere resolved by SDS-PAGE and transferred to a polyvinylidenedifluoride membrane. The membrane was blocked with 5% nonfatmilk for 1 h, followed by incubation with rabbit anti-nephrinantiserum UP3 (1:3000 dilution) (11) for another 1 h at roomtemperature. Nephrin bands were visualized by chemiluminescenceusing secondary antibodies labeled with horseradish peroxidase.
Co-localization Experiments
Cells stably expressing wild-type or mutant nephrin were culturedto 60 to 80% confluence and then incubated for 24 h at 37°Cin the presence or absence of 4-PBA. After that, the cells weretrypsinized, plated onto 10-cm plates, and cultured overnight.They were then transfected with a modified pCDM0 vector containinga cDNA encoding the extracellular domain of Neph1 fused withthe human IgG Fc-portion (Neph1.ec.fc; provided by Dr. Gerke,University of Freiburg, Freiburg, Germany). After transfection,the cells were plated on glass coverslips and stained for Nephrin-Fcand nephrin. For staining the Neph1-Fc-chimera, FITC-conjugatedpolyclonal anti-human IgG was used (dilution 1:200). Nephrinwas stained using the monoclonal anti-nephrin antibody 50A9.Secondary antibodies used were TRITC-conjugated anti-rabbitIgG and FITC-conjugated anti-mouse IgG (Dako).
Antibody Cross-Linking
Near-confluent cell cultures were washed once with ice-coldDMEM containing 25 mM HEPES (pH 7.4) and then incubated withthe 50A9 monoclonal anti-nephrin antibody (10 µg/ml inDMEM, containing 25 mM HEPES [pH 7.4]) for 30 min at 4°C.After removing the primary antibody solution, the cells wereincubated for 1 min at 37°C with 20 µg/ml goat anti-mouseIgG (Jackson ImmunoResearch Laboratories, West Grove, PA) inDMEM containing 25 mM HEPES (pH 7.4). The secondary antibodieswere removed, and the plates were rinsed gently once with PBS.The cells were lysed either in the SDS–sample buffer orin a buffer containing 0.5% sodium deoxycholate, 0.1% SDS, 10mM sodium fluoride, 1 mM sodium orthovanadate, and 1 mM PMSF(RIPA-buffer). The former buffer was used when analyzing totalcell lysates, and the RIPA buffer was used when nephrin wasfirst immunoprecipitated from the lysates. For immunoprecipitation,we used a mixture of the mAb 43H7 and 43C7 (12). The sampleswere analyzed by SDS-PAGE and Western blotting using the anti-phosphotyrosineand reprobed with anti-nephrin antibodies.
Chemical Chaperone Rescues Nephrin Missense Mutants from ER to the Plasma Membrane
Restoration of impaired trafficking of disease-causing mutantproteins has recently become an interesting experimental andtherapeutic concept. Successful rescue of proteins, such asCFTR and 1-antitrypsin mutants in cell culture models, is welldocumented (9,10). For investigating whether 4-PBA can alterthe cellular localization of nephrin missense mutants, 16 mutantsthat are not transported from the ER to cell membrane (Table 1)(8) were expressed in the presence of 10 mM 4-PBA. As shownin Table 1 and Figure 1, wild-type nephrin was well expressedon the plasma membrane when cultured in the absence (Figure 1A)or presence (Figure 1B) of 4-PBA. In contrast, dramaticdifferences were observed in the case of some of the nephrinmissense mutants. As shown in Table 1 and Figure 1, C, E, G, I, and K,none of the 16 mutants was expressed on the cell surfacewhen cultured in the absence of 4-PBA. However, when grown inthe presence of 10 mM 4-PBA for 24 h, six of the mutants wererescued from the ER to the cell surface (Table 1, Figure 1, D, J, and L;immunofluorescence microscopy data not shown forthe mutants R367C, R802W, and R831C).
Figure 1. Sodium 4-phenylbutyrate (4-PBA) rescues some of the nephrin mutants to the cell surface. Stable HEK293 transfectants expressing either wild-type nephrin or the different mutant forms were grown in the absence or presence of 4-PBA, and then stained with anti-nephrin mAb 50A9 (FITC) without prior permeabilization. This mAb recognizes the eighth Ig motif of nephrin. Filamentous actin was stained with rhodamine-conjugated phalloidin. Wild-type nephrin is expressed on the cell surface no matter whether 4-PBA is present or not (A and B). None of the mutants grown in the absence of 4-PBA expresses nephrin on the cell surface (C, E, G, I, and K). However, of the 16 nephrin mutants studied (Table 1), 6 were found to be rescued to the cell surface by 4-PBA. Here, we show the stainings for the mutants W64S, S350P, and S366R (D, J, and L).
Cell Surface Biotinylation and Western Blot Analysis
To verify the results obtained by immunofluorescence microscopy,we performed cell surface biotinylation and Western blot analysis.In Figure 2, we show the results for the nephrin missense mutantswhose immunolocalization is shown in Figure 1. Of these fivemutants, three (W64S, S350P, and S366R) were rescued by 4-PBAto the cell surface, whereas the mutants 1171N and G270C wereretained inside the cells. In line with these data, when cellsgrown in the presence of 4-PBA were biotinylated with the membrane-impermeablebiotinylation reagent sulfo-NHS-biotin, only the mutants W64S,S350P, and S366R were labeled (Figure 2, top). As expected,none of the mutants but only wild-type nephrin was labeled whenthe cells were grown in the absence of 4-PBA. Importantly, analysisof the total cell lysates indicated that the tested nephrinforms were expressed at equal levels (Figure 2, bottom). Itis not shown here, but the results from the biotinylation experimentfor the rest of the mutants listed in Table 1 were also fullyin line with the immunofluorescence microscopy data. Therefore,the results presented in Table 1 and Figures 1 and 2 convincinglydemonstrated the ability of 4-PBA to restore the cell surfaceexpression of some of the nephrin mutants.
Figure 2. Examination of cell surface expression with the use of a membrane-impermeable biotinylation reagent. The different transfectants grown in the absence or presence of 4-PBA were incubated on ice with sulfo-NHS-biotin, after which biotinylated proteins were captured by streptavidin-agarose and analyzed by Western blotting using an antibody recognizing the cytoplasmic domain of nephrin (UP3). The upper panel indicates the presence of nephrin on the plasma membrane (PM) in case of the nephrin forms whose immunolocalization is show in Figure 1. Wild-type nephrin (cell line NPH15) was found to be expressed on the cell surface in cells grown either in the absence or presence of 4-PBA. In line with the immunofluorescence data (Figure 1), the mutants W64S, S350P, and S366R were rescued to the cell surface by 4-PBA. The lower panel represents total cell lysates probed with the anti-nephrin antibody.
Rescued Nephrin Mutants Interact with Slit Diaphragm Partner Neph1
To investigate whether the rescued nephrin mutants were biologicallyfunctional, we tested their interaction with Neph1, anotherprotein of the slit diaphragm. We chose to study this interactionbecause it is, besides the nephrin–nephrin interaction,so far the only interaction assigned for the extracellular domainof nephrin (13–15). To this end, a plasmid encoding theextracellular part of Neph1 tagged with the human IgG Fc-portionwas transfected into normal 293 cells or into the cells stablyexpressing wild-type nephrin, the six mutant forms found tobe rescued by 4-PBA, and also into cells expressing one of themutants (I171N) that was not rescued by 4-PBA. As expected,FITC-labeled anti-human IgG did not stain normal unpermeabilized293 cells transfected with the plasmid encoding Neph1 or thestable I171N mutants transfected with the Neph1-plasmid (Figure 3, A and E),yet the transfection was successful, because astrong signal was detected in Triton-permeabilized cells (Figure 3, B and F).In contrast, when transfectants expressing Neph1and wild-type nephrin or the six rescued mutants were stainedfor Neph1 without permeabilization, an abundant signal was detected.In Figure 3, the results for wild-type nephrin and the mutantW64S are shown (Figure 3, C and G). Double staining furtherconfirmed that wild-type nephrin and the W64S mutant co-localizedwith Neph1 on the cell surface (Figure 4). The other nephrinmutants rescued by 4-PBA—S350P, S366R, R367C, R802W, andR831C—also co-localized with Neph1 on the plasma membrane(data not shown).
Figure 3. Wild-type nephrin and the rescued mutants associate with the soluble Neph1-Fc fusion protein on the cell surface. A plasmid encoding a soluble version of the transmembrane protein Neph1, Neph1-Fc, was transfected into normal 293 cells as well as into the cells expressing the different forms of nephrin. The cells were cultured in the presence of 4-PBA, and then stained for Neph1-Fc (FITC) without (A, C, E, and G) or with (B, D, F, and H) before permeabilization. With regard to the rescued mutants, the results only for the mutant W64S are shown here. This mutant (G) as well as wild-type nephrin (C) were found to be interacting with Neph1-Fc on the cell surface. No cell surface expression of Neph1-Fc was detected in the cells expressing only Neph1-Fc (A), or both Neph1-Fc and the nephrin mutant form I171N (E). Panels B, D, F, and H demonstrate that the plasmid encoding Neph1-Fc was successfully transfected into all tested cell lines. Filamentous actin was stained with rhodamine-conjugated phalloidin.
Figure 4. Double staining for nephrin (FITC) and Neph1-Fc (rhodamine) in the transfectants expressing both Neph1-Fc and either wild-type nephrin or the rescued mutant W64S. The merged pictures indicate that Neph1-Fc colocalizes with both forms of nephrin.
Antibody Cross-Linking Triggers Signaling from the Rescued Mutants
Nephrin is a signaling molecule phosphorylated by Src familykinases (16–18). To examine whether the rescued mutantsare able to initiate signaling, we clustered nephrin on thesurface of the transfectants by the anti-nephrin mAb 50A9. Asshown in Figure 5, clustering induced strong and rapid nephrintyrosine phosphorylation in the cells expressing either wild-typenephrin or the mutant W64S cultured in the presence of 4-PBA.In contrast, no nephrin tyrosine phosphorylation was detectedin the same clones when the cells were treated with a controlantibody or when the W64S mutant grown in the absence of 4-PBAwas clustered. Furthermore, the clustering procedure did notinduce any nephrin tyrosine phosphorylation in the cells expressingthe nonrescued mutant I171N grown in the presence of 4-PBA.In Figure 5, the results from two separate experiments are shown.In panels A and B, the total cell lysates and nephrin immunoprecipitates,respectively, were analyzed by Western blotting with anti-phosphotyrosineand anti-nephrin antibodies. The other rescued nephrin mutants—S350P,S366R, R367C, R802W, and R831C—behaved identically tothe mutant W64S (data not shown).
Figure 5. Clustering of nephrin on the surface of the wild-type transfectants or the transfectants expressing the rescued mutants induces robust nephrin tyrosine phosphorylation. Subconfluent cultures of the cells expressing the different nephrin forms were first incubated with the anti-nephrin mAb 50A9 on ice for 30 minutes, and nephrin was then clustered at 37°C with goat anti-mouse IgG for 1 min. (A) Cells were lysed in the SDS-PAGE sample buffer containing phosphatase inhibitors and analyzed by SDS-PAGE and Western blotting with anti-phosphotyrosine (upper panel) and anti-nephrin antibodies (lower panel). (B) Samples were analyzed in the same manner after first immunoprecipitating nephrin from the cell lysates. With regard to the rescued mutants, the results only for the mutant W64S are shown. Clustering induces strong nephrin tyrosine phosphorylation in the cells expressing wild-type nephrin or the mutant W64S grown in the presence of 4-PBA.
In the cell, the ER has a quality control system for "proofreading"newly synthesized proteins, ensuring that only correctly foldedproteins with native conformation reach their final destination.The incompletely assembled and misfolded proteins are retainedin the ER and finally degraded (19–21). Not surprising,a number of human genetic diseases are caused by ER retentionand degradation of folding-defective mutant proteins (22–24).It seems that a number of CNF patients belong to this category,too (8). Curious is that >30 different missense mutationsdescribed in CNF patients result in the severe null-like phenotype(7). We showed previously in a cell culture study that the molecularpathomechanism underlying a severe phenotype is likely to involveretention of misfolded nephrin mutants in the ER, resultingin complete loss of nephrin from the cell surface (8).
During recent years, 4-PBA has been shown effectively to rescuevarious mutated proteins that are arrested in ER and allow themto be expressed on cell surface and also be correctly functional,such as CFTR in cystic fibrosis and 1-antitrypsin in emphysemaand liver disease (9,10). 4-PBA is a known oral administer regularlyused as an ammonia scavenger in the treatment of urea cycledisorders (25). It has already been tested in clinical trialswith patients with cystic fibrosis. These trials are based onthe observation that 4-PBA promotes the trafficking of the F508-CFTRmutant to the cell surface (9,26). The mechanism of the 4-PBAeffect is not yet completely clear, but it acts at least asa transcriptional regulator of proteins involved in the ER qualitycontrol system (27,28). In this study, we observed that 4-PBAcould prevent retention of several mutant nephrin proteins inthe ER and promote their transport to the cell surface. However,4-PBA did not rescue the cell surface expression of all of thenephrin mutants, which may reflect different degrees of impairmentin the folding of these proteins. Recently, Neph1 was identifiedas another important component of the slit diaphragm, whereit forms cis hetero-oligomers with nephrin (13–15). Inaddition to being a structural component of the slit diaphragm,nephrin is a signaling protein that is heavily tyrosine phosphorylatedwhen dislocated from the slit diaphragm or cross-linked withanti-nephrin antibodies (17,29). Nephrin signaling is augmentedby podocin, a protein that has been shown to be an intracellularinteraction partner of nephrin and target nephrin into lipidrafts (16,30). All of the rescued nephrin mutants studied herewere found to interact with Neph1 on cell surface, and theirtyrosine phosphorylation was rapidly induced by clustering,suggesting that the rescued mutant nephrins could be functional.Consequently, this study suggests that the use of 4-PBA or correspondingchemical chaperones may provide a potential future therapeuticapproach for CNF. It is interesting that very recently a point-mutatedpodocin protein was shown to be rescued to the plasma membranein a cell culture system by treatment with chemical chaperonestrimethylamine-N-oxide and DMSO (31). Podocin is mutated inan autosomal recessive steroid-resistant nephrotic syndromeand in some patients with a nonfamilial focal segmental glomerulosclerosis(32,33). These are indeed more ideal candidates for the treatmentwith pharmacologic chaperones than CNF patients, because CNFis manifested strongly already during fetal development in utero.In CNF, the most probable use and benefit of chemical chaperoneswould be in the treatment of newborn patients during the periodof intensive care to slow down the progression of nephroticsyndrome.
Acknowledgments
This work was supported in part by grants from the Novo NordiskFoundation and the Swedish Medical Research Council.
We are grateful to Dr. Gerke (University of Freiburg, Freiburg,Germany) for kindly providing the Neph1.ec.Fc plasmid, VesaRuotsalainen for anti-nephrin antibodies, and Jamshid Khoshnoodifor valuable discussions.
Hallman N, Norio R, Kouvalainen K: Main features of the congenital nephrotic syndrome. Acta Paediatr Scand Suppl 172: 175–178, 1967
Holmberg C, Laine J, Ronnholm K, Ala-Houhala M, Jalanko H: Congenital nephrotic syndrome. Kidney Int Suppl 53: S51–S56, 1996[Medline]
Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K: Positionally cloned gene for a novel glomerular protein—nephrin—is mutated in congenital nephrotic syndrome. Mol Cell 1: 575–582, 1998[CrossRef][Medline]
Ruotsalainen V, Ljungberg P, Wartiovaara J, Lenkkeri U, Kestila M, Jalanko H, Holmberg C, Tryggvason K: Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proc Natl Acad Sci U S A 96: 7962–7967, 1999[Abstract/Free Full Text]
Beltcheva O, Martin P, Lenkkeri U, Tryggvason K: Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome. Hum Mutat 17: 368–373, 2001[CrossRef][Medline]
Gigante M, Monno F, Roberto R, Laforgia N, Assael MB, Livolti S, Caringella A, La Manna A, Masella L, Iolascon A: Congenital nephrotic syndrome of the Finnish type in Italy: A molecular approach. J Nephrol 15: 696–702, 2002[Medline]
Koziell A, Grech V, Hussain S, Lee G, Lenkkeri U, Tryggvason K, Scambler P: Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration. Hum Mol Genet 11: 379–388, 2002[Abstract/Free Full Text]
Liu L, Done SC, Khoshnoodi J, Bertorello A, Wartiovaara J, Berggren PO, Tryggvason K: Defective nephrin trafficking caused by missense mutations in the NPHS1 gene: Insight into the mechanisms of congenital nephrotic syndrome. Hum Mol Genet 10: 2637–2644, 2001[Abstract/Free Full Text]
Rubenstein RC, Egan ME, Zeitlin PL: In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR. J Clin Invest 100: 2457–2465, 1997[Medline]
Burrows JA, Willis LK, Perlmutter DH: Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. Proc Natl Acad Sci U S A 97: 1796–1801, 2000[Abstract/Free Full Text]
Ruotsalainen V, Patrakka J, Tissari P, Reponen P, Hess M, Kestila M, Holmberg C, Salonen R, Heikinheimo M, Wartiovaara J, Tryggvason K, Jalanko H: Role of nephrin in cell junction formation in human nephrogenesis. Am J Pathol 157: 1905–1916, 2000[Abstract/Free Full Text]
Ruotsalainen V, Reponen P, Khoshnoodi J, Kilpeläinen P, Tryggvason K: Monoclonal antibodies to human nephrin. Hybrid Hybridomics 23: 55–63, 2004[CrossRef][Medline]
Barletta GM, Kovari IA, Verma RK, Kerjaschki D, Holzman LB: Nephrin and Neph1 co-localize at the podocyte foot process intercellular junction and form cis hetero-oligomers. J Biol Chem 278: 19266–19271, 2003[Abstract/Free Full Text]
Gerke P, Huber TB, Sellin L, Benzing T, Walz G: Homodimerization and heterodimerization of the glomerular podocyte proteins nephrin and NEPH1. J Am Soc Nephrol 14: 918–926, 2003[Abstract/Free Full Text]
Liu G, Kaw B, Kurfis J, Rahmanuddin S, Kanwar YS, Chugh SS: Neph1 and nephrin interaction in the slit diaphragm is an important determinant of glomerular permeability. J Clin Invest 112: 209–221, 2003[CrossRef][Medline]
Huber TB, Kottgen M, Schilling B, Walz G, Benzig T: Interaction with podocin facilitates nephrin signaling. J Biol Chem 276: 41543–41546, 2001[Abstract/Free Full Text]
Lahdenpera J, Kilpelainen P, Liu XL, Pikkarainen T, Reponen P, Ruotsalainen V, Tryggvason K: Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases. Kidney Int 64: 404–413, 2003[CrossRef][Medline]
Huber TB, Hartleben B, Kim J, Schmidts M, Schermer B, Keil A, Egger L, Lecha RL, Borner C, Pavenstadt H, Shaw AS, Walz G, Benzing T: Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling. Mol Cell Biol 23: 4917–4928, 2003[Abstract/Free Full Text]
Fewell SW, Travers KJ, Weissman JS, Brodsky JL: The action of molecular chaperones in the early secretory pathway. Annu Rev Genet 35: 149–191, 2001[CrossRef][Medline]
Ellgaard L, Helenius A: ER quality control: Towards an understanding at the molecular level. Curr Opin Cell Biol 13: 431–437, 2001[CrossRef][Medline]
Ellgaard L, Helenius A: Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4: 181–191, 2003[CrossRef][Medline]
Thomas PJ, Qu BH, Pedersen PL: Defective protein folding as a basis of human disease. Trends Biochem Sci 20: 456–459, 1995[CrossRef][Medline]
Kuznetsov G, Nigam SK: Folding of secretory and membrane proteins. N Engl J Med 339: 1688–1695, 1998[Free Full Text]
Bross P, Corydon TJ, Andresen BS, Jorgensen MM, Bolund L, Gregersen N: Protein misfolding and degradation in genetic diseases. Hum Mutat 14: 186–198, 1999[CrossRef][Medline]
Brusilow SW: Phenylacetylglutamine may replace urea as a vehicle for waste nitrogen excretion. Pediatr Res 29: 147–150, 1991
Zeitlin PL, Diener-West M, Rubenstein RC, Boyle MP, Lee CK, Brass-Ernst L: Evidence of CFTR function in cystic fibrosis after systemic administration of 4-phenylbutyrate. Mol Ther 6: 119–126, 2002[CrossRef][Medline]
Rubenstein RC, Lyons BM: Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation. Am J Physiol Lung Cell Mol Physiol 281: L43–L51, 2001[Abstract/Free Full Text]
Simons M, Schwartz K, Kriz W, Miettinen A, Reiser J, Mundel P, Holthofer H: Involvement of lipids rafts in nephrin phosphorylation and organization of the glomerular slit diaphragm. Am J Pathol 159: 1069–1077, 2001[Abstract/Free Full Text]
Huber TB, Simons M, Hartleben B, Sernetz L, Schmidts M, Gundlach E, Saleem MA, Walz G, Benzig T: Molecular basis of the functional podocin-nephrin complex: Mutations in NPHS2 gene disrupt nephrin targeting to lipid raft microdomains. Hum Mol Genet 12: 3397–3405, 2003[Abstract/Free Full Text]
Ohashi T, Uchida K, Uchida S, Sasaki S, Nihei H: Intracellular mislocalization of mutant podocin and correction by chemical chaperons. Histochem Cell Biol 119: 257–264, 2003[CrossRef][Medline]
Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler MC, Niaudet P, Antignac C: NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 24: 349–354, 2000[CrossRef][Medline]
Caridi G, Bertelli R, Carrea A, Di Duca M, Catarsi P, Artero M, Carraro M, Zennaro C, Candiano G, Musante L, Seri M, Ginevri F, Perfumo F, Ghiggeri GM: Prevalence, genetics, and clinical features of patients carrying podocin mutations in steroid-resistant nonfamilial focal segmental glomerulosclerosis. J Am Soc Nephrol 12: 2742–2746, 2001[Abstract/Free Full Text]
Received for publication November 5, 2003.
Accepted for publication April 8, 2004.
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Science,
February 15, 2008;
319(5865):
916 - 919.
[Abstract][Full Text][PDF]
N. Cheong, M. Madesh, L. W. Gonzales, M. Zhao, K. Yu, P. L. Ballard, and H. Shuman Functional and Trafficking Defects in ATP Binding Cassette A3 Mutants Associated with Respiratory Distress Syndrome
J. Biol. Chem.,
April 7, 2006;
281(14):
9791 - 9800.
[Abstract][Full Text][PDF]
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Abstract
Introduction
1-antitrypsin (9,10). In this study, we examined the potential effect of 4-PBA on the trafficking of nephrin missense mutants by using immunofluorescence microscopy, cell surface biotinylation, and Western blotting. It was demonstrated that 4-PBA is able to rescue a number of the nephrin missense mutants to escape the ER quality control. The proteins are transported to the plasma membrane, where they function indistinguishably from wild-type nephrin, as judged by their ability to interact with Neph1 and to become heavily tyrosine phosphorylated when clustered by anti-nephrin antibodies. 
convincingly demonstrated the ability of 4-PBA to restore the cell surface expression of some of the nephrin mutants. 
F508-CFTR mutant to the cell surface (9,26). The mechanism of the 4-PBA effect is not yet completely clear, but it acts at least as a transcriptional regulator of proteins involved in the ER quality control system (27,28). In this study, we observed that 4-PBA could prevent retention of several mutant nephrin proteins in the ER and promote their transport to the cell surface. However, 4-PBA did not rescue the cell surface expression of all of the nephrin mutants, which may reflect different degrees of impairment in the folding of these proteins. Recently, Neph1 was identified as another important component of the slit diaphragm, where it forms cis hetero-oligomers with nephrin (13–15). In addition to being a structural component of the slit diaphragm, nephrin is a signaling protein that is heavily tyrosine phosphorylated when dislocated from the slit diaphragm or cross-linked with anti-nephrin antibodies (17,29). Nephrin signaling is augmented by podocin, a protein that has been shown to be an intracellular interaction partner of nephrin and target nephrin into lipid rafts (16,30). All of the rescued nephrin mutants studied here were found to interact with Neph1 on cell surface, and their tyrosine phosphorylation was rapidly induced by clustering, suggesting that the rescued mutant nephrins could be functional. Consequently, this study suggests that the use of 4-PBA or corresponding chemical chaperones may provide a potential future therapeutic approach for CNF. It is interesting that very recently a point-mutated podocin protein was shown to be rescued to the plasma membrane in a cell culture system by treatment with chemical chaperones trimethylamine-N-oxide and DMSO (31). Podocin is mutated in an autosomal recessive steroid-resistant nephrotic syndrome and in some patients with a nonfamilial focal segmental glomerulosclerosis (32,33). These are indeed more ideal candidates for the treatment with pharmacologic chaperones than CNF patients, because CNF is manifested strongly already during fetal development in utero. In CNF, the most probable use and benefit of chemical chaperones would be in the treatment of newborn patients during the period of intensive care to slow down the progression of nephrotic syndrome. 
