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CFTR Folding Consortium: Methods Available for Studies of CFTR Folding and Correction

  • Kathryn W. Peters
  • Tsukasa Okiyoneda
  • William E. Balch
  • Ineke Braakman
  • Jeffrey L. Brodsky
  • William B. Guggino
  • Christopher M. Penland
  • Harvey B. Pollard
  • Eric J. Sorscher
  • William R. Skach
  • Philip J. Thomas
  • Gergely L. Lukacs
  • Raymond A. Frizzell
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 742)

Abstract

The CFTR Folding Consortium (CFC) was formed in 2004 under the auspices of the Cystic Fibrosis Foundation and its drug discovery and development affiliate, CFF Therapeutics. A primary goal of the CFC is the development and distribution of reagents and assay methods designed to better understand the mechanistic basis of mutant CFTR misfolding and to identify targets whose manipulation may correct CFTR folding defects. As such, reagents available from the CFC primarily target wild-type CFTR NBD1 and its common variant, F508del, and they include antibodies, cell lines, constructs, and proteins. These reagents are summarized here, and two protocols are described for the detection of cell surface CFTR: (a) an assay of the density of expressed HA-tagged CFTR by ELISA and (b) the generation and use of an antibody to CFTR’s first extracellular loop for the detection of endogenous CFTR. Finally, we highlight a systematic collection of assays, the CFC Roadmap, which is being used to assess the cellular locus and mechanism of mutant CFTR correction. The Roadmap queries CFTR structure–function relations at levels ranging from purified protein to well-differentiated human airway primary cultures.

Key words

Protein folding protein degradation antibody generation cell surface protein detection research consortium www.cftrfolding.org 

Notes

Acknowledgments

Resources providing support for this work in the Frizzell lab include grants from the NIH (DK068196 and DK 072506) and the Cystic Fibrosis Foundation (CFF R883-CR07 and FRIZZE05XX0). Experimental work in the laboratory of Gergely Lukacs was funded by the NIH, Cystic Fibrosis Folding Consortium, CIHR, and CFI. Tsukasa Okiyoneda was supported by a postdoctoral fellowship from the Canadian Cystic Fibrosis Foundation.

References

  1. 1.
    Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., et al. (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63, 827–834.PubMedCrossRefGoogle Scholar
  2. 2.
    Zhang, F., Kartner, N., and Lukacs, G. L. (1998) Limited proteolysis as a probe for arrested conformational maturation of delta F508 CFTR. Nat Struct Biol 5, 180–183.PubMedCrossRefGoogle Scholar
  3. 3.
    Du, K., Sharma, M., and Lukacs, G. L. (2005) The DeltaF508 cystic fibrosis mutation impairs domain-domain interactions and arrests post-translational folding of CFTR. Nat Struct Mol Biol 12, 17–25.PubMedCrossRefGoogle Scholar
  4. 4.
    Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., Smith, A. E., and Welsh, M. J. (1992) Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358, 761–764.PubMedCrossRefGoogle Scholar
  5. 5.
    Welch, W. J. (2004) Role of quality control pathways in human diseases involving protein misfolding. Semin Cell Dev Biol 15, 31–38.PubMedCrossRefGoogle Scholar
  6. 6.
    Wang, X., Venable, J., LaPointe, P., Hutt, D. M., Koulov, A. V., Coppinger, J., et al. (2006) Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 127, 803–815.PubMedCrossRefGoogle Scholar
  7. 7.
    Zhang, Y., Nijbroek, G., Sullivan, M. L., McCracken, A. A., Watkins, S. C., Michaelis, S., et al. (2001) Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Mol Biol Cell 12, 1303–1314.PubMedGoogle Scholar
  8. 8.
    Sun, F., Mi, Z., Condliffe, S. B., Bertrand, C. A., Gong, X., Lu, X., et al. (2008) Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia. FASEB J 22, 3255–3263.PubMedCrossRefGoogle Scholar
  9. 9.
    DeCarvalho, A. C., Gansheroff, L. J., and Teem, J. L. (2002) Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta F508. J Biol Chem 277, 35896–35905.PubMedCrossRefGoogle Scholar
  10. 10.
    Teem, J. L., Carson, M. R., and Welsh, M. J. (1996) Mutation of R555 in CFTR-delta F508 enhances function and partially corrects defective processing. Receptors Channels 4, 63–72.PubMedGoogle Scholar
  11. 11.
    Chang, X. B., Cui, L., Hou, Y. X., Jensen, T. J., Aleksandrov, A. A., Mengos, A., et al. (1999) Removal of multiple arginine-framed trafficking signals overcomes misprocessing of delta F508 CFTR present in most patients with cystic fibrosis. Mol Cell 4, 137–142.PubMedCrossRefGoogle Scholar
  12. 12.
    Pedemonte, N., Lukacs, G. L., Du, K., Caci, E., Zegarra-Moran, O., Galietta, L. J., et al. (2005) Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening. J Clin Invest 115, 2564–2571.PubMedCrossRefGoogle Scholar
  13. 13.
    Van Goor, F., Straley, K. S., Cao, D., Gonzalez, J., Hadida, S., Hazlewood, A., et al. (2006) Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules. Am J Physiol Lung Cell Mol Physiol 290, L1117–L1130.PubMedCrossRefGoogle Scholar
  14. 14.
    Pyle, L. C., Balch, W. E., Lukacs, G., Braakman, I., Guggino, W. B., Thomas, P. J., et al. (2010) Developing a cellular road map for correctors of protein misfolding: a consortium approach. Nat Rev Drug Disc. (submitted).Google Scholar
  15. 15.
    Lukacs, G. L., Segal, G., Kartner, N., Grinstein, S., and Zhang, F. (1997) Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation. Biochem J 328 (Pt 2), 353–361.PubMedGoogle Scholar
  16. 16.
    Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., et al. (1999) Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal. J Biol Chem 274, 3602–3609.PubMedCrossRefGoogle Scholar
  17. 17.
    Benharouga, M., Haardt, M., Kartner, N., and Lukacs, G. L. (2001) COOH-terminal truncations promote proteasome-dependent degradation of mature cystic fibrosis transmembrane conductance regulator from post-Golgi compartments. J Cell Biol 153, 957–970.PubMedCrossRefGoogle Scholar
  18. 18.
    Denning, G. M., Ostedgaard, L. S., Cheng, S. H., Smith, A. E., and Welsh, M. J. (1992) Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia. J Clin Invest 89, 339–349.PubMedCrossRefGoogle Scholar
  19. 19.
    Denning, G. M., Ostedgaard, L. S., and Welsh, M. J. (1992) Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia. J Cell Biol 118, 551–559.PubMedCrossRefGoogle Scholar
  20. 20.
    Sharma, M., Pampinella, F., Nemes, C., Benharouga, M., So, J., Du, K., et al. (2004) Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes. J Cell Biol 164, 923–933.PubMedCrossRefGoogle Scholar
  21. 21.
    Glozman, R., Okiyoneda, T., Mulvihill, C. M., Rini, J. M., Barriere, H., and Lukacs, G. L. (2009) N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic. J Cell Biol 184, 847–862.PubMedCrossRefGoogle Scholar
  22. 22.
    Barriere, H., Bagdany, M., Bossard, F., Okiyoneda, T., Wojewodka, G., Gruenert, D., et al. (2009) Revisiting the role of cystic fibrosis transmembrane conductance regulator and counterion permeability in the pH regulation of endocytic organelles. Mol Biol Cell 20, 3125–3141.PubMedCrossRefGoogle Scholar
  23. 23.
    Robert, R., Carlile, G. W., Pavel, C., Liu, N., Anjos, S. M., Liao, J., et al. (2008) Structural analog of sildenafil identified as a novel corrector of the F508del-CFTR trafficking defect. Mol Pharm 73, 478–489.Google Scholar
  24. 24.
    Carlile, G. W., Robert, R., Zhang, D., Teske, K. A., Luo, Y., Hanrahan, J. W., et al. (2007) Correctors of protein trafficking defects identified by a novel high-throughput screening assay. ChemBioChem 8, 1012–1020.PubMedCrossRefGoogle Scholar
  25. 25.
    Voller, A., Bidwell, D., Rose, N. R., Friedman, H., and Fahey, J. L. (1986) Enzyme-linked immunosorbent assay, in Manual of Clinical Laboratory Immunology, vol. 3. American Society of Microbiology, Washington, DC, pp. 99–109.Google Scholar
  26. 26.
    Harlow, E., and Lane, D. (1988) Immunoprecipitation, in Antibodies: A Practical Approach. Google Scholar
  27. 27.
    Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1073.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Kathryn W. Peters
    • 1
  • Tsukasa Okiyoneda
    • 2
  • William E. Balch
    • 3
  • Ineke Braakman
    • 4
  • Jeffrey L. Brodsky
    • 5
  • William B. Guggino
    • 6
  • Christopher M. Penland
    • 7
  • Harvey B. Pollard
    • 8
  • Eric J. Sorscher
    • 9
  • William R. Skach
    • 10
  • Philip J. Thomas
    • 11
  • Gergely L. Lukacs
    • 2
  • Raymond A. Frizzell
    • 12
  1. 1.Department of Cell Biology and PhysiologyUniversity of PittsburghPittsburghUSA
  2. 2.Department of PhysiologyMcGill UniversityMontrealCanada
  3. 3.Department of Cell BiologyThe Scripps Research InstituteLa JollaUSA
  4. 4.Cellular Protein ChemistryUtrecht UniversityUtrechtThe Netherlands
  5. 5.Department of Biological SciencesUniversity of PittsburghPittsburghUSA
  6. 6.Department of PhysiologyJohns Hopkins UniversityBaltimoreUSA
  7. 7.Cystic Fibrosis FoundationBethesdaUSA
  8. 8.Department of Anatomy, Physiology, and GeneticsUniformed Services University of the Health SciencesBethesdaUSA
  9. 9.Gregory Fleming James Cystic Fibrosis Research CenterUniversity of Alabama at BirminghamBirminghamUSA
  10. 10.Department of Biochemistry and Molecular BiologyOregon Health & Science UniversityPortlandUSA
  11. 11.Department of PhysiologyUniversity of Texas Southwestern Medical CenterDallasUSA
  12. 12.Department of Cell Biology and PhysiologyUniversity of Pittsburgh School of MedicinePittsburghUSA

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