Abstract
Defective protein folding is becoming increasingly recognized as a significant cause of human disease, and cystic fibrosis (CF) is a prime example. A number of CF-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in a CFTR protein that does not reach the plasma membrane but is instead retained by the cellular quality control system and degraded by the ubiquitin-proteasome system. Misfolded CFTR that cannot be degraded accumulates in the cell as centrosome-associated inclusions of aggregated protein that are replete with proteasome components. In fact, the centrosomal region is a significant site of proteasome concentration in resting cells, suggesting a novel role for this subcellular location in the quality control of protein expression. This chapter gives an overview of CFTR misfolding, degradation, and aggregation, and provides details of methods used in our laboratory to study these processes.
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Riordan, J. R., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Iannuzzi, M. C., Collins, F. S., and Tsui, L.-C. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245 1066–1073.
Anderson, M. P., Gregory, R. J., Thompson, S., Souza, D. W., Paul, S., Mulligan, R. C., Smith, A. E., and Welsh, M. J. (1991) Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253, 202–205.
Bear, C. E., Duguay, F., Naismith, A. L., Kartner, N., Hanrahan, J. W., and Riordan, J. R. (1991) Cl-channel activity in Xenopus oocytes expressing the cystic fibrosis gene. J. Biol. Chem. 266, 19,142–19,145.
Sheppard, D. N. and Welsh, M. J. (1999) Structure and function of the CFTR chloride channel. Physiol. Rev. 79, S23–S45.
Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999) Regulation of Cl-/HCO3-exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells. J. Biol. Chem. 274, 3414–3421.
Schwiebert, E. M., Benos, D. J., Egan, M. E., Stutts, M. J., and Guggino, W. B. (1999) CFTR is a conductance regulator as well as a chloride channel. Physiol. Rev. 79[1], S145–S166.
Thomas, P. J., Ko, Y. H., and Pedersen, P. L. (1992) Altered protein folding may be the molecular basis of most cases of cystic fibrosis. FEBS Lett. 312, 7–9.
Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., O’Riordan, C. R., and Smith, A. E. (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63, 827–834.
Lukacs, G. L., Mohamed, A., Kartner, N., Chang, X.-B., Riordan, J. R., and Grinstein, S. (1994) Conformational maturation of CFTR but not its mutant counterpart (F508) occurs in the endoplasmic reticulum and requires ATP. EMBO J. 13, 6076–6086.
Ward, C. L. and Kopito, R. R. (1994) Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. J. Biol. Chem. 269, 25,710–25,718.
Yang, Y., Janich, S., Cohn, J. A., and Wilson, J. M. (1993) The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment. Proc. Natl. Acad. Sci. USA 90, 9480–9484.
Strickland, E., Qu, B.-H., Millen, L., and Thomas, P. (1997) The molecular chaperone Hsc70 assists the in vitro folding of the N-terminal nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 272, 25,421–25,424.
Meacham, G. C., Lu, Z., King, S., Sorscher, E., Tousson, A., and Cyr, D. M. (1999) The Hdj-2Hsc70 chaperone pair facilitates early steps in CFTR biogenesis. EMBO J. 18, 1492–1505.
Loo, M. A., Jensen, T. J., Cui, L., Hou, Y.-X., Chang, X.-B., and Riordan, J. R. (1998) Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome. EMBO J. 17, 6879–6887.
Pind, S., Riordan, J. R., and Williams, D. B. (1994) Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 269, 12,784–12,788.
Ellgaard, L., Molinari, M., and Helenius, A. (1999) Setting the standards: quality control in the secretory pathway. Science 286, 1882–1888.
Wigley, W. C., Fabunmi, R. P., Lee, M. G., Marino, C. R., Muallem, S., DeMartino, G. N., and Thomas, P. J. (1999) Dynamic association of proteasomal machinery with the centrosome. J. Cell Biol. 145, 481–490.
Gilbert, A., Jadot, M., Leontieva, E., Wattiaux-De Coninck, S., and Wattiaux, R. (1998) F508 CFTR localizes in the endoplasmic reticulum-golgi intermediate compartment in cystic fibrosis cells. Exp. Cell. Res. 242, 144–152.
Ward, C. L., Omura, S., and Kopito, R. R. (1995) Degragation of CFTR by the ubiquitin-proteasome pathway. Cell 83, 121–127.
Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83, 129–135.
Coux, O., Tanaka, K., and Goldberg, A. L. (1996) Structure and functions of the 20S and 26S proteasomes. Annu. Rev. Biochem. 65, 801–847.
Baumeister, W., Walz, J., Zuhl, F., and Seemuller, E. (1998) The proteasome: paradigm of a self-compartmentalizing protease. Cell 92, 367–380.
Wenzel, T. and Baumeister, W. (1995) Conformational constraints in protein degradation by the 20S proteasome. Nat. Struct. Biol. 2, 199–204.
Lowe, J., Stock, D., Jap, B., Zwickl, P., Baumeister, W., and Huber, R. (1995) Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 268, 533–539.
Groll, M., Ditzel, L., Lowe, J., Stock, D., Bochtler, M., Bartunik, H. D., and Huber, R. (1997) Structure of 20S proteasome from yeast at 2. 4 A resolution. Nature 386, 463–471.
Yoshimura, T., Kameyama, K., Takagi, T., Ikai, A., Tokunaga, F., Koide, T., Tanahashi, N., Tamura, T., Cejka, Z., Baumeister, W., Tanaka, K., and Ichihara, A. (1993) Molecular characterization of the ÉC;26SÉD; proteasome complex from rat liver. J. Struct. Biol. 111, 200–211.
Adams, G. M., Falke, S., Goldberg, A. L., Slaughter, C. A., DeMartino, G. N., and Gogol, E. P. (1997) Structural and functional effects of PA700 and modulator protein on proteasomes. J. Mol. Biol. 273, 646–657.
Hochstrasser, M. (1996) Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30, 405–439.
Ciechanover, A. (1994) The ubiquitin-proteasome proteolytic pathway Cell 79, 13–21.
Lam, Y. A., Xu, W., DeMartino, G. N., and Cohen, R. E. (1997) Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 385, 737–740.
Ma, C.-P., Vu, J. H., Proske, R. J., Slaughter, C. A., and DeMartino, G. N. (1994) Identification, purification, and characterization of a high molecular weight ATP-dependent activator (PA700) of the 20S proteasome. J. Biol. Chem. 269, 3539–3547.
Gray, C. W., Slaughter, C. A., and DeMartino, G. N. (1994) PA28 activator protein forms regulatory caps on proteasome stacked rings. J. Mol. Biol. 236, 7–15.
Dubiel, W., Pratt, G., Ferrell, K., and Rechsteiner, M. (1992) Purification of an 11S regulator of the multicatalytic protease. J. Biol. Chem. 267, 22,369–22,377.
Ma, C.-P., Slaughter, C. A., and DeMartino, G. N. (1992) Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain) J. Biol. Chem. 267, 10,515–10,523.
Groettrup, M., Soza, A., Eggers. M., Kuehn, L., Dick, T. P., Schild, H., Rammensee, H.-G., Koszinowski, U. H., and Kloetzel, P.-M. (1996) A role for the proteasome regulator PA28a in antigen presentation. Nature 381, 166–168.
Kloetzel, P. M., Soza, A., and Stohwasser, R. (1999) The role of the proteasome system and the proteasome activator PA28 complex in the cellular immune response. Biol. Chem. 380, 293–297.
Xiong, X., Chong, E., and Skach, W. R. (1999) Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane. J. Biol. Chem. 274, 2616–2624.
Sato, S., Ward, C. L., and Kopito, R. R. (1998) Cotranslational ubiquitination of cystic fibrosis transmembrane conductance regulator in vitro. J. Biol. Chem. 273, 7189–7192.
Plemper, R. K. and Wolf, D. H. (1999) Retrograde protein translocation: ERADication of secretory proteins in health and disease. Trends Biochem. Sci. 24, 266–270.
Plemper, R. K. and Wolf, D. H. (1999) Endoplasmic reticulum degradation. Reverse protein transport and its end in the proteasome. Mol. Biol. Rep. 26, 125–130.
Sommer, T. and Wolf, D. H. (1997) Endoplasmic reticulum degradation: reverse protein flow of no return. FASEB J. 11, 1227–1233.
Bebok, Z., Mazzochi, C., King, S. A., Hong, J. S., and Sorscher, E. J. (1998) The mechanism underlying cystic fibrosis transmembrane conductance regulator transport from the endoplasmic reticulum to the proteasome includes Sec61beta and a cytosolic deglycosylated intermediary. J. Biol. Chem. 273, 29,873–29,878.
Xiong, X., Bragin, A., Widdicombe, J. H., Cohn, J., and Skach, W. R. (1997) Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. J. Clin. Invest 100, 1079–1088.
Plemper, R. K., Deak, P. M., Otto, R. T., and Wolf, D. H. (1999) Re-entering the translocon from the lumenal side of the endoplasmic reticulum. Studies on mutated carboxypeptidase yscY species. FEBS Lett. 443, 241–245.
Plemper, R. K., Egner, R., Kuchler, K., and Wolf, D. H. (1998) Endoplasmic reticulum degradation of a mutated ATP-binding cassette transporter Pdr5 proceeds in a concerted action of Sec61 and the proteasome. J. Biol. Chem. 273, 32,848–32,856.
Wickner, S., Maurizi, M. R., and Gottesman, S. (1999) Posttranslational quality control: folding, refolding, and degrading proteins. Science 286, 1888–1893.
Agashe, V. R. and Hartl, F. U. (2000) Roles of molecular chaperones in cytoplasmic protein folding. Semin. Cell Dev. Biol. 11, 15–25.
Thomas, P. J., Qu, B.-H., and Pedersen, P. L. (1995) Defective protein folding as a basis of human disease. TIBS 20, 456–459.
Gottesman, S., Wickner, S., and Maurizi, M. R. (1997) Protein quality control: triage by chaperones and proteases. Genes Dev. 11, 815–823.
Bercovich, B., Stancovski, I., Mayer, A., Blumenfeld, N., Laszlo, A., Schwartz, A., and Ciechanover, A. (1997) Ubiquitin-dependent degradation of certain protein substrates in vitro requires the molecular chaperone Hsc70. JBC 272, 9002–9010.
Strickland, E., Hakala, K., Thomas, P. J., and DeMartino, G. N. (2000) Recognition of misfolding proteins by PA700, the regulatory subcomplex of the 26 S proteasome. J. Biol. Chem. 275, 5565–5572.
Johnston, J. A., Ward, C. L., and Kopito, R. R. (1998) Aggresomes: A Cellular Response to Misfolded Proteins. J. Cell Biol. 143, 1883–1898.
Lange, B. M., Bachi, A., Wilm, M., and Gonzalez, C. (2000) Hsp90 is a core centrosomal component and is required at different stages of the centrosome cycle in Drosophila and vertebrates. EMBO J. 19, 1252–1262.
Brown, C. R., Doxsey, S. J., Hong-Brown, L. Q., Martin, R. L., and Welch, W. J. (1996) Molecular chaperones and the centrosome. A role for TCP-1 in microtubule nucleation. J. Biol. Chem. 271, 824–832.
Brown, C. R., Hong-Brown, L. Q., Doxsey, S. J., and Welch, W. J. (1996) Molecular chaperones and the centrosome. A role for HSP 73 in centrosomal repair following heat shock treatment. J. Biol. Chem. 271, 833–840.
Chen, E. Y., Bartlett, M. C., and Clarke, D. M. (2000) Cystic fibrosis transmembrane conductance regulator has an altered structure when its maturation is inhibited. Biochemistry 39, 3797–3803.
Fabunmi, R. P., Wigley, W. C., Thomas, P. J., and DeMartino, G. N. (2000) Activity and regulation of the centrosome-associated proteasome. J. Biol. Chem. 275, 409–413.
Doxsey, S. J. (1998) The centrosome-a tiny organelle with big potential. Nature Genetics 20, 104–106.
Zimmerman, W., Sparks, C. A., and Doxsey, S. J. (1999) Amourphous no longer: the centrosome comes into focus. Curr. Opin. Cell Biol. 11, 122–128.
Urbani, L. and Stearns, T. (1999) The centrosome. Curr. Biol. 9, R315–R317.
Koepp, D. M., Harper, J. W., and Eledge, S. J. (1999) How the Cyclin Became a Cyclin: Tegulated Proteolysis in the Cell Cycle. Cell 97, 431–434.
Bailly, E., Pines, J., Hunter, T., and Bornens, M. (1992) Cytoplasmic accumulation of cyclin B1 in human cells: association with a detergent-resistant compartment and with the centrosome. J. Cell. Sci. 101, 529–545.
Brown, C. R., Doxsey, S. J., White, E., and Welch, W. J. (1994) Both viral (adenovirus E1B) and cellular (hsp70, p53) components interact with centrosomes. J. Cell. Physiol. 160, 47–60.
Crepieux, P., Kwon, H., Leclerc, N., Spencer, W., Richard, S., Lin, R., and Hiscott, J. (1997) IKBa physically interacts with a cytoskeleton-associated protein through its signal response domain. Mol. Cell. Biol. 17, 7375–7385.
Zeng, W., Lee, M. G., Yan, M., Diaz, J., Benjamin, I., Marino, C. R., Kopito, R., Freedman, S., Cotton, C., Muallem, S., and Thomas, P. (1997) Immuno and functional characterization of CFTR in submandibular and pancreatic acinar and duct cells. Am. J. Physiol. 273, C442–C455.
Ma, C.-P., Willy, P. J., Slaughter, C. A., and DeMartino, G. N. (1993) PA28, an activator of the 20s proteasome, is inactivated by proteolytic modification of its carboxyl terminus. J. Biol. Chem. 268, 22,514–22,519.
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Corboy, M.J., Thomas, P.J., Wigley, W.C. (2002). CFTR Degradation and Aggregation. In: Skach, W.R. (eds) Cystic Fibrosis Methods and Protocols. Methods in Molecular Medicine™, vol 70. Humana Press. https://doi.org/10.1385/1-59259-187-6:277
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DOI: https://doi.org/10.1385/1-59259-187-6:277
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