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- W2017230110 abstract "PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated ΔF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL·CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis. PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated ΔF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL·CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis. Loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) 2The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PDZ domain, PSD-95, Discs-large, Zonula occludens-1 domain; NHERF, Na+/H+ exchanger regulatory factor; CAL, CFTR-associated ligand; BCRP, breast cancer resistance protein; DTT, dithiothreitol; CFTRinh-172, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone; GST, glutathione S-transferase; SEC, size exclusion chromatography; HSQC, heteronuclear single quantum correlation spectroscopy; WT, wild type; siRNA, small interfering RNA; PBS, phosphate-buffered saline; CV, column volumes; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HA, hemagglutinin; WCL, whole-cell lysate. 2The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PDZ domain, PSD-95, Discs-large, Zonula occludens-1 domain; NHERF, Na+/H+ exchanger regulatory factor; CAL, CFTR-associated ligand; BCRP, breast cancer resistance protein; DTT, dithiothreitol; CFTRinh-172, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone; GST, glutathione S-transferase; SEC, size exclusion chromatography; HSQC, heteronuclear single quantum correlation spectroscopy; WT, wild type; siRNA, small interfering RNA; PBS, phosphate-buffered saline; CV, column volumes; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HA, hemagglutinin; WCL, whole-cell lysate. are the underlying cause of cystic fibrosis (1Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5907) Google Scholar, 2Rommens J.M. Iannuzzi M.C. Kerem B. Drumm M.L. Melmer G. Dean M. Rozmahel R. Cole J.L. Kennedy D. Hidaka N. Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2525) Google Scholar). CFTR forms ATP-gated Cl- channels in the apical membrane of epithelial cells in a variety of tissues. In the lung, it plays an essential role in regulating the fluid and ion balance required for the correct function of mucociliary clearance mechanisms (3Wine J.J. J. Clin. Investig. 1999; 103: 309-312Crossref PubMed Scopus (214) Google Scholar). Genetic analysis has revealed over 1400 distinct disease-associated CFTR mutations, which exhibit widely varying effects at the molecular level. Some lead to the complete loss of ion channel function. Others, however, retain at least partial chloride channel conductivity, but lead to incorrect folding and/or intracellular trafficking of the protein, such that the mutant CFTR does not reach the apical membrane (4Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1416) Google Scholar, 5Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 358: 761-764Crossref PubMed Scopus (1057) Google Scholar). This applies in particular to the most common genetic lesion associated with CF, in which the codon for Phe508 is deleted (ΔF508) (6Fuller C.M. Benos D.J. Am. J. Physiol. 1992; 263: C267-C286Crossref PubMed Google Scholar, 7Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1224) Google Scholar). Even for WT CFTR, a large fraction of newly synthesized protein is degraded before reaching the apical membrane (8Kopito R.R. Physiol. Rev. 1999; 79: S167-173Crossref PubMed Scopus (374) Google Scholar), and the protein that does is subjected to continual endocytosis and endocytic recycling (9Bradbury N.A. Cohn J.A. Venglarik C.J. Bridges R.J. J. Biol. Chem. 1994; 269: 8296-8302Abstract Full Text PDF PubMed Google Scholar, 10Prince L.S. Peter K. Hatton S.R. Zaliauskiene L. Cotlin L.F. Clancy J.P. Marchase R.B. Collawn J.F. J. Biol. Chem. 1999; 274: 3602-3609Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). As a result, regulation of CFTR intracellular trafficking is important for its function in both physiological and pathological contexts. Genetic, biochemical, and cell biological studies have revealed a complex network of protein-protein interactions that are required for correct CFTR trafficking, including a number of PDZ (PSD-95, discs-large, zonula occludens-1) proteins, which act as adaptor molecules, coupling CFTR to other components of the trafficking and localization machinery, and to other transmembrane channels and receptors (11Kunzelmann K. News Physiol. Sci. 2001; 16: 167-170PubMed Google Scholar, 12Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 426-436Crossref PubMed Scopus (346) Google Scholar). Class I PDZ domains typically recognize C-terminal binding motifs characterized by the sequence–(S/T)-X-Φ-COOH (where Φ represents a hydrophobic side chain, and X represents any amino acid) (13Harris B.Z. Lim W.A. J. Cell Sci. 2001; 114: 3219-3231Crossref PubMed Google Scholar, 14Brône B. Eggermont J. Am. J. Physiol. 2005; 288: C20-C29Crossref PubMed Scopus (86) Google Scholar). The cytoplasmic C terminus of CFTR satisfies the class I PDZ binding motif, ending in the sequence -DTRL (15Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman N. Welsh M.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (375) Google Scholar, 16Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 17Wang S.S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (249) Google Scholar). Earlier work in some of our laboratories had shown that the CFTR C-terminal PDZ-binding motif controls retention of the protein at the apical membrane and modulates its endocytic recycling (18Moyer B.D. Duhaime M. Shaw C. Denton J. Reynolds D. Karlson K.H. Pfeiffer J. Wang S.S. Mickle J.E. Milewski M. Cutting G.R. Guggino W.B. Li M. Stanton B.A. J. Biol. Chem. 2000; 275: 27069-27074Abstract Full Text Full Text PDF PubMed Google Scholar, 19Swiatecka-Urban A. Duhaime M. Coutermarsh B. Karlson K.H. Collawn J. Milewski M. Cutting G.R. Guggino W.B. Langford G. Stanton B.A. J. Biol. Chem. 2002; 277: 40099-40105Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). PDZ proteins that have been shown to interact with CFTR include NHERF1 (Na+/H+ exchanger regulatory factor 1; also known as EBP50), NHERF2 (aka E3KARP), NHERF3 (aka CAP70, PDZK1, or NaPi CAP-1), NHERF4 (aka IKEPP or NaPi CAP-2), and CAL (CFTR-associated ligand; aka PIST, GOPC, and FIG) (12Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 426-436Crossref PubMed Scopus (346) Google Scholar, 20Li C. Naren A.P. Pharmacol. Ther. 2005; 108: 208-223Crossref PubMed Scopus (117) Google Scholar). Overexpression of CAL in heterologous cells leads to a dramatic decrease in the plasma-membrane levels of CFTR (21Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) and of several other membrane proteins that are known to interact with it, including Clc-3 chloride channels, the β1 adrenergic receptor, and the somatostatin receptor subtype 5 (22Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 23He J. Bellini M. Xu J. Castleberry A.M. Hall R.A. J. Biol. Chem. 2004; 279: 50190-50196Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 24Wente W. Stroh T. Beaudet A. Richter D. Kreienkamp H.J. J. Biol. Chem. 2005; 280: 32419-32425Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In the case of CFTR, the effect is mediated by reductions in the rate of membrane insertion and in the half-life of the channels at the cell surface (21Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), and can be prevented by blocking endocytosis or lysosomal degradation (25Cheng J. Wang H. Guggino W.B. J. Biol. Chem. 2004; 279: 1892-1898Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The negative effect of CAL overexpression on CFTR expression levels can also be reversed by the simultaneous overexpression of NHERF1, which competes for the C-terminal TRL binding motif (21Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), or by overexpression of TC10, a Rho GTPase whose constitutively active form redistributes CAL intracellularly toward the plasma membrane (26Cheng J. Wang H. Guggino W.B. J. Biol. Chem. 2005; 280: 3731-3739Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). It thus appears that CAL plays an important role in the intracellular trafficking and localization of CFTR. Furthermore, because high levels of CAL reduce CFTR levels, it is possible that endogenous CAL acts as a negative regulator. If so, targeted modulation of the CAL-CFTR interaction could provide a mechanism for up-regulating CFTR trafficking in a therapeutic context, in analogy to the rescue of ΔF508-CFTR seen upon overexpression of NHERF1 (27Guerra L. Fanelli T. Favia M. Riccardi S.M. Busco G. Cardone R.A. Carrabino S. Weinman E.J. Reshkin S.J. Conese M. Casavola V. J. Biol. Chem. 2005; 280: 40925-40933Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). However, previous studies have focused on the effects of CAL overexpression on WT-CFTR. As a result, no evidence has been available as to whether endogenous CAL is limiting for CFTR expression nor whether its effects apply to disease-associated mutants. Furthermore, analysis of the regulatory interactions has so far been confined to heterologous cells, even though trafficking pathways depend strongly on cellular context (28Yoshimori T. Keller P. Roth M.G. Simons K. J. Cell Biol. 1996; 133: 247-256Crossref PubMed Scopus (201) Google Scholar, 29Tuma P.L. Nyasae L.K. Hubbard A.L. Mol. Biol. Cell. 2002; 13: 3400-3415Crossref PubMed Scopus (43) Google Scholar, 30Swiatecka-Urban A. Brown A. Moreau-Marquis S. Renuka J. Coutermarsh B. Barnaby R. Karlson K.H. Flotte T.R. Fukuda M. Langford G.M. Stanton B.A. J. Biol. Chem. 2005; 280: 36762-36772Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In the experiments reported here, we test the hypothesis that suppression of endogenous CAL expression levels will increase the cell-surface expression of functional ΔF508-CFTR and that it will do so in a polarized human bronchial epithelial cell line. In addition, we assess the ability of a localized mutational knock-out of the CAL PDZ binding pocket to abrogate CAL-mediated suppression of cell-surface CFTR, providing new insights into the mechanism of interaction. Taken together, our results establish the potential therapeutic relevance of pharmaceutical inhibition of the CAL PDZ binding domain. siRNA-mediated Targeting of Endogenous CAL Expression—CFBE41o- cells (31Bruscia E. Sangiuolo F. Sinibaldi P. Goncz K.K. Novelli G. Gruenert D.C. Gene Ther. 2002; 9: 683-685Crossref PubMed Scopus (131) Google Scholar, 32Gruenert D.C. Willems M. Cassiman J.J. Frizzell R.A. J. Cyst. Fibros. 2004; 3 (Suppl. 2): 191-196Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) stably transduced with the ΔF508-CFTR gene under control of a cytomegalovirus promoter (“CFBE+ΔF508” cells) (33Li Y. Wang W. Parker W. Clancy J.P. Am. J. Respir. Cell Mol. Biol. 2006; 34: 600-608Crossref PubMed Scopus (39) Google Scholar) were a generous gift of Dr. J. P. Clancy (University of Alabama, Birmingham) and were maintained in the Dartmouth CF Core Facility. Monolayers of CFBE+ΔF508 cells were grown in 6-well plates and transfected with 160 nm CAL-specific siRNA (GOPC3; Qiagen) or nonspecific siRNA (control, non-silencing siRNA; Qiagen) or an equal volume of medium, using the transfection reagent Lipofectamine 2000 (Invitrogen). After 20 h, cells were provided with fresh medium. To measure cell-surface CFTR, 72 h after transfection, cells were washed with ice-cold phosphate-buffered saline (Invitrogen), incubated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce; 1 mg/ml in phosphate-buffered saline with 1 mm MgCl2, 0.1 mm CaCl2, pH 8.2) for 1 h at 4 °C, washed, lysed in lysis buffer (25 mm HEPES, pH 8.2, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 Complete tablet/50 ml (Roche)), collected using a cell scraper (Sarstedt), and centrifuged. An aliquot of clarified whole-cell lysate (WCL) was subjected to SDS-PAGE and analyzed by Western blotting with CFTR-, CAL-, and ezrin-specific antibodies. The remaining clarified WCL was incubated with streptavidin beads overnight at 4 °C, after which the beads were washed three times with lysis buffer. Proteins were eluted in Laemmli sample buffer/dithiothreitol (DTT) at 85 °C for 5 min, and resolved by SDS-PAGE. Western blotting was performed with antibodies specific for CFTR, breast cancer resistance protein (BCRP), and the Na+/K+-ATPase α1 subunit. Horseradish perioxidase-conjugated secondary antibody (Bio-Rad) and Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) were used for visualization. For experiments with polarized monolayers, CFBE+ΔF508 cells were seeded at low density. For biochemical experiments, 105 cells were seeded on 24-mm diameter Transwell filters (Corning) and allowed to grow for 3 days prior to transfection. For electrophysiological experiments 3.3 × 104 cells were seeded on 12-mm diameter Snapwell filters (Corning), and allowed to grow for 4 days prior to transfection. In both cases, subconfluent monolayers were transfected overnight with 50 nm CAL-specific or nonspecific siRNA (GOPC3 or control, non-silencing siRNA, respectively; Qiagen), using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s protocol. Confluent monolayers were allowed to form, and cells were serum-starved for 24 h, and switched to 27 °C for 24–36 h prior to experimentation to increase signal intensity. Monolayers were apically biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce), and WCL and surface-biotinylated samples were prepared and analyzed as described above for non-polarized cells. Electrophysiology—Seven days after seeding monolayers, Ussing chamber measurements were performed essentially as described (34Swiatecka-Urban A. Moreau-Marquis S. Maceachran D.P. Connolly J.P. Stanton C.R. Su J.R. Barnaby R. O'Toole G.A. Stanton B.A. Am. J. Physiol. 2006; 290: C862-C872Crossref PubMed Scopus (59) Google Scholar), except that 50 μm amiloride was used. For these studies, 50 μm genistein was applied apically to activate temperature-rescued ΔF508-CFTR channels (34Swiatecka-Urban A. Moreau-Marquis S. Maceachran D.P. Connolly J.P. Stanton C.R. Su J.R. Barnaby R. O'Toole G.A. Stanton B.A. Am. J. Physiol. 2006; 290: C862-C872Crossref PubMed Scopus (59) Google Scholar, 35Bebok Z. Collawn J.F. Wakefield J. Parker W. Li Y. Varga K. Sorscher E.J. Clancy J.P. J. Physiol. 2005; 569: 601-615Crossref PubMed Scopus (148) Google Scholar). Once maximal activation was achieved, 5 μm 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTRinh-172, EMD Biosciences, Refs. 36Ma T. Thiagarajah J.R. Yang H. Sonawane N.D. Folli C. Galietta L.J. Verkman A.S. J. Clin. Investig. 2002; 110: 1651-1658Crossref PubMed Scopus (584) Google Scholar and 37Taddei A. Folli C. Zegarra-Moran O. Fanen P. Verkman A.S. Galietta L.J. FEBS Lett. 2004; 558: 52-56Crossref PubMed Scopus (95) Google Scholar) was applied apically to inhibit CFTR-mediated chloride currents. Data are reported as the difference between the genistein-activated and the CFTRinh-172-inhibited short-circuit currents (Isc). Recombinant Protein Expression Vectors—Full-length human CAL (GenBank™ accession AF450008; TrEMBL accession number Q969U8) was subcloned into the pET16b expression vector (Novagen) on an NdeI/BamHI fragment generated by PCR to yield the vector pHCAL1. The 5′ primer was designed to introduce a decahistidine purification tag at the N terminus of the construct. The CAL PDZ domain (amino acids 278–362) was also PCR subcloned into pET16b as an NdeI/BamHI fragment to yield the vector pHCALP5. Its 5′ primer was designed to introduce an N-terminal decahistidine tag followed by a TEV protease recognition sequence. CAL-binding site mutants were prepared using the QuikChange and Multichange protocols (Stratagene) in the eukaryotic expression vector pECFP-CAL, containing full-length CAL inserted as an EcoRI/BamHI fragment into the pECFP-C1 backbone (Clontech): “CAL-D” = S294D,T296E,K340D,K342E; “CAL-E” = K299D,K340D,K342E; and “CAL-T+L” = L291E,G292E,I295E,H341F,L348N. Full-length and PDZ domain mutant constructs were subcloned into the bacterial expression vectors described above and into the mammalian expression vector encoding HA-tagged full-length CAL (25Cheng J. Wang H. Guggino W.B. J. Biol. Chem. 2004; 279: 1892-1898Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). pGST-CFTRC, encoding C-terminal residues 1377–1480 of CFTR as a glutathione S-transferase (GST) fusion protein in the pGEX-4T-1 vector (GE Healthcare), was obtained from the Dartmouth Cystic Fibrosis Core Facility and was originally a generous gift of Drs. P. Devarajan and A. Swiatecka-Urban. pGST-CFTRCΔTRL was PCR subcloned as a BamHI/SalI site fragment into pGEX-4T-1. All protein expression constructs were verified by DNA sequencing. Protein Expression—pHCAL1-transformed BL21(DE3) RIL cells (Novagen) were grown at 37 °C in LB medium to an A600 of ∼0.6. Protein expression was induced with 0.1 mm isopropyl β-d-thiogalactopyranoside and allowed to proceed for 16 h at 20 °C. Cells were harvested, resuspended in lysis buffer T (50 mm Tris, pH 8.5, 150 mm NaCl, 10% (w/v) glycerol, 1 mm DTT, 0.1 mm ATP, 25 units/ml benzonase (EMD Biosciences), 2 mm MgCl2, supplemented with one EDTA-free Complete tablet per 50 ml) and lysed using a French press. pHCALP5-transformed BL21(DE3) RIL cells were grown at 37 °C in 2× YT medium to an A600 of ∼0.8. Induction, expression, and lysis conditions were identical to those for full-length CAL, except that the lysis buffer did not contain glycerol. Isotopically labeled CAL PDZ protein was expressed for NMR analysis in 15N M9 minimal media (including 1× BME vitamins (Sigma), 4 mg/liter thiamine HCl (Sigma), and 1% (w/v) glucose). 15NH4Cl was obtained from Spectra Stable Isotopes or Cambridge Isotope Laboratories. Mutant CAL and CAL-PDZ proteins were expressed under the same conditions as wild-type proteins. pGEX-4T-1-, pGST-CFTRC-, and pGST-CFTRCΔTRL-transformed BL21(DE3) cells were grown at 37 °C in LB medium to an A600 of ≥0.6. Protein expression was induced by addition of 0.5 mm isopropyl β-d-thiogalactopyranoside and allowed to proceed overnight at 20 °C. Cells were harvested, resuspended in lysis buffer ((phosphate-buffered saline (PBS): 137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 2 mm KH2PO4, pH 7.3), 1 mg/ml lysozyme, 10 μg/ml DNase I (Roche), 5 mm DTT, 5 mm MgSO4, supplemented with 1 Complete tablet in 50 ml). After incubation for 30 min on ice, the cells were lysed using a French press. Protein Purification—All lysates were clarified by centrifugation at 40,000 rpm in a Ti45 rotor for 1 h at 4 °C. Imidazole was added to the CAL-PDZ supernatants to a final concentration of 10 mm before application to a nickel-nitrilotriacetic acid Super-flow (Qiagen) column (bed volume 10 ml), which had been pre-equilibrated with 5 column volumes (CV) of TBS-CAL (50 mm Tris, pH 8.5, 150 mm NaCl, 1 mm DTT, 0.1 mm ATP), containing 10 mm imidazole. Following sample application, the column was washed with 10 CV of TBS-CAL containing 10 mm imidazole, and protein was eluted in TBS-CAL with a linear gradient of 10–400 mm imidazole over 20 CV. Eluates were collected in tubes containing Chelex 100 Molecular Biology grade resin (Bio-Rad). CAL was purified using a similar protocol, except that TBS-CAL was supplemented for metal affinity chromatography with 10% (w/v) glycerol and 0.1% (w/v) Triton X-100. CAL- or CAL-PDZ-containing fractions were pooled, centrifuged at 3700 × g for 10 min, and filtered through a 0.45-μm polyvinylidene difluoride filter (Millipore) to remove any residual Chelex resin. GST-CFTRC fusion proteins were purified by affinity chromatography using glutathione-Sepharose 4 Fast Flow beads (Sigma) (bed volume 12 ml). The column was equilibrated with 3 CV of PBS containing 0.05% (v/v) Tween 20 (ICN; PBS/Tween). Following sample application, the column was washed with 5 CV of PBS/Tween and the fusion protein eluted with 4 CV of PBS containing 25 mm glutathione. The pooled eluates were applied to HiLoad Superdex 200 (CAL; GST-CFTRC) or Superdex 75 (CAL-PDZ) prep grade 16/60 or 26/60 size-exclusion chromatography (SEC) columns (GE Healthcare) in TBS-CAL containing 0.02% sodium azide and 25 mm, instead of 50 mm Tris (CAL; CAL-PDZ) or in PBS/Tween (GST-CFTR fusions). The purity of all proteins was assessed by SDS-PAGE. CAL was concentrated in Amicon Ultra-15 10,000 MWCO, CAL-PDZ in Centricon Plus 80 Biomax-5 5,000 MWCO, and GST-CFTR fusion proteins in Amicon Ultra-15 30,000 MWCO concentrators (Millipore). Following concentration, the oligomeric homogeneity of CAL and CAL-PDZ proteins was verified by analytical SEC. Pull-down Binding Assay—Pull-down experiments were carried out by directly mixing the two proteins under a given interaction condition described below. 500 μl of glutathione-Sepharose bead slurry (Sigma) was aliquoted into an Eppendorf tube. After a brief centrifugation (1,000 × g; 1 min), the liquid above the beads was carefully aspirated. The beads were equilibrated twice with 1 ml each of PBS/Tween. An aliquot containing 200 μg of GST or GST fusion protein was added (after removal of residual glutathione using a PD10 desalting column; GE Healthcare), and the volume adjusted to 1 ml with the same buffer. The mixture was incubated on ice for 1 h with shaking every 10 min to permit GST capture. After centrifuging the tubes for 5 min at 1000 × g, unbound material was discarded, and the beads were washed thoroughly. An aliquot containing 200 μg of CAL or CAL-PDZ proteins was added to the captured GST or GST fusion protein, and the volume adjusted to 1 ml. The interaction was allowed to proceed for 1 h on ice with shaking every 10 min. After complex formation was completed, to remove unbound protein, the beads were repeatedly washed until the supernatant contained no protein as detected using Bradford reagent. The washed beads were resuspended with an equal volume of SDS-PAGE loading buffer, boiled for 3 min at 95 °C, and bound proteins visualized by SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining. Homology Modeling—Homology modeling was performed via the web-based SWISS-MODEL server (38Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4445) Google Scholar). In one case, the program selected the templates automatically (Protein Data Bank entries 1QAV and 2PDZ, both syntrophin; 1UEZ, KIAA1526 PDZ1; 1UF1, KIAA1526 PDZ2; 1BE9, PSD-95 PDZ3). In a second case, the program was provided with a user-defined template (Protein Data Bank entry 1I92) corresponding to the NHERF1-PDZ1-CFTRC crystal structure (39Karthikeyan S. Leung T. Ladias J.A. J. Biol. Chem. 2001; 276: 19683-19686Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The latter model was combined with the CFTR C-terminal peptide contributed by a symmetry-related molecule in the crystal lattice to generate the model of the CAL·CFTR complex shown in Fig. 4. Least-squares superpositions were performed using the program LSQKAB (40Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2326) Google Scholar). Structural representations were prepared using the program MOLSCRIPT (41Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar). Mass Spectrometry and NMR Analysis of CAL Protein and Mutants—Following SEC purification, wild-type CAL, CAL-D, and CAL-E mutants and the corresponding PDZ domain proteins were subjected to MALDI-TOF analysis in the Dartmouth Molecular Biology & Proteomics Core Facility. The CAL-PDZ-D mutant domain was also subjected to 1H,15N-heteronuclear single quantum correlation spectroscopy (HSQC) analysis, as described (42Piserchio A. Fellows A. Madden D.R. Mierke D.F. Biochemistry. 2005; 44: 16158-16166Crossref PubMed Scopus (19) Google Scholar). Assays of CFTR Expression in the Presence of CAL-binding Site Mutants—A GFP-CFTR fusion protein was expressed in African green monkey kidney (COS-7) cells in the presence or absence of wild-type and mutant HA-CAL. Both proteins were detected by Western blotting as previously described (25Cheng J. Wang H. Guggino W.B. J. Biol. Chem. 2004; 279: 1892-1898Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Endogenous CAL Down-regulates ΔF508-CFTR Cell-Surface Expression—Our previous studies had shown that overexpression of CAL in heterologous cell lines reduces the levels of recombinant WT-CFTR found in whole cell lysates and at the cell surface. This effect could be blocked by the overexpression of NHERF1 together with CAL (21Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Recently, overexpression of NHERF1 has been shown to rescue the cell-surface expression of ΔF508-CFTR in a human bronchial epithelial cell line (27Guerra L. Fanelli T. Favia M. Riccardi S.M. Busco G. Cardone R.A. Carrabino S. Weinman E.J. Reshkin S.J. Conese M. Casavola V. J. Biol. Chem. 2005; 280: 40925-40933Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Given the apparent antagonism of CAL and NHERF1, we suspected that reduction of endogenous CAL exp" @default.
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- W2017230110 title "Targeting CAL as a Negative Regulator of ΔF508-CFTR Cell-Surface Expression" @default.
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