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. 1997 Mar 15;99(6):1432–1444. doi: 10.1172/JCI119302

Correcting temperature-sensitive protein folding defects.

C R Brown 1, L Q Hong-Brown 1, W J Welch 1
PMCID: PMC507959  PMID: 9077553

Abstract

Recently, we found that different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in correcting the temperature-sensitive protein folding defect associated with the deltaF508 cystic fibrosis transmembrane regulator (CFTR) protein. Here we examined whether the folding of other proteins which exhibit temperature-sensitive folding defects also could be corrected via a similar strategy. Cell lines expressing temperature-sensitive mutants of the tumor suppressor protein p53, the viral oncogene protein pp60src, or a ubiquitin activating enzyme E1, were incubated at the nonpermissive temperature (39.5 degrees C) in the presence of glycerol, trimethylamine N-oxide or deuterated water. In each case, the cells exhibited phenotypes similar to those observed when the cells were incubated at the permissive temperature (32.5 degrees C), indicative that the particular protein folding defect had been corrected. These observations, coupled with our earlier work and much older studies in yeast and bacteria, indicate that protein stabilizing agents are effective in vivo for correcting protein folding abnormalities. We suggest that this type of approach may prove to be useful for correcting certain protein folding abnormalities associated with human diseases.

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Selected References

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  1. Back J. F., Oakenfull D., Smith M. B. Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry. 1979 Nov 13;18(23):5191–5196. doi: 10.1021/bi00590a025. [DOI] [PubMed] [Google Scholar]
  2. Brown C. R., Hong-Brown L. Q., Biwersi J., Verkman A. S., Welch W. J. Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones. 1996 Jun;1(2):117–125. doi: 10.1379/1466-1268(1996)001<0117:ccctmp>2.3.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cheng S. H., Gregory R. J., Marshall J., Paul S., Souza D. W., White G. A., O'Riordan C. R., Smith A. E. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell. 1990 Nov 16;63(4):827–834. doi: 10.1016/0092-8674(90)90148-8. [DOI] [PubMed] [Google Scholar]
  4. Chowdary D. R., Dermody J. J., Jha K. K., Ozer H. L. Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway. Mol Cell Biol. 1994 Mar;14(3):1997–2003. doi: 10.1128/mcb.14.3.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Denning G. M., Anderson M. P., Amara J. F., Marshall J., Smith A. E., Welsh M. J. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature. 1992 Aug 27;358(6389):761–764. doi: 10.1038/358761a0. [DOI] [PubMed] [Google Scholar]
  6. Drumm M. L., Wilkinson D. J., Smit L. S., Worrell R. T., Strong T. V., Frizzell R. A., Dawson D. C., Collins F. S. Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes. Science. 1991 Dec 20;254(5039):1797–1799. doi: 10.1126/science.1722350. [DOI] [PubMed] [Google Scholar]
  7. Edington B. V., Whelan S. A., Hightower L. E. Inhibition of heat shock (stress) protein induction by deuterium oxide and glycerol: additional support for the abnormal protein hypothesis of induction. J Cell Physiol. 1989 May;139(2):219–228. doi: 10.1002/jcp.1041390202. [DOI] [PubMed] [Google Scholar]
  8. Finlay C. A., Hinds P. W., Tan T. H., Eliyahu D., Oren M., Levine A. J. Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life. Mol Cell Biol. 1988 Feb;8(2):531–539. doi: 10.1128/mcb.8.2.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gannon J. V., Lane D. P. Protein synthesis required to anchor a mutant p53 protein which is temperature-sensitive for nuclear transport. Nature. 1991 Feb 28;349(6312):802–806. doi: 10.1038/349802a0. [DOI] [PubMed] [Google Scholar]
  10. Gekko K., Koga S. Increased thermal stability of collagen in the presence of sugars and polyols. J Biochem. 1983 Jul;94(1):199–205. doi: 10.1093/oxfordjournals.jbchem.a134330. [DOI] [PubMed] [Google Scholar]
  11. Gekko K., Timasheff S. N. Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. Biochemistry. 1981 Aug 4;20(16):4667–4676. doi: 10.1021/bi00519a023. [DOI] [PubMed] [Google Scholar]
  12. Gekko K., Timasheff S. N. Thermodynamic and kinetic examination of protein stabilization by glycerol. Biochemistry. 1981 Aug 4;20(16):4677–4686. doi: 10.1021/bi00519a024. [DOI] [PubMed] [Google Scholar]
  13. HAWTHORNE D. C., FRIIS J. OSMOTIC-REMEDIAL MUTANTS. A NEW CLASSIFICATION FOR NUTRITIONAL MUTANTS IN YEAST. Genetics. 1964 Nov;50:829–839. doi: 10.1093/genetics/50.5.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hawkes R., Grutter M. G., Schellman J. Thermodynamic stability and point mutations of bacteriophage T4 lysozyme. J Mol Biol. 1984 May 15;175(2):195–212. doi: 10.1016/0022-2836(84)90474-1. [DOI] [PubMed] [Google Scholar]
  15. Henle K. J., Peck J. W., Higashikubo R. Protection against heat-induced cell killing by polyols in vitro. Cancer Res. 1983 Apr;43(4):1624–1627. [PubMed] [Google Scholar]
  16. Kulka R. G., Raboy B., Schuster R., Parag H. A., Diamond G., Ciechanover A., Marcus M. A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1. J Biol Chem. 1988 Oct 25;263(30):15726–15731. [PubMed] [Google Scholar]
  17. Li C., Ramjeesingh M., Reyes E., Jensen T., Chang X., Rommens J. M., Bear C. E. The cystic fibrosis mutation (delta F508) does not influence the chloride channel activity of CFTR. Nat Genet. 1993 Apr;3(4):311–316. doi: 10.1038/ng0493-311. [DOI] [PubMed] [Google Scholar]
  18. Lin P. S., Kwock L., Hefter K. Protection of heat induced cytoxicity by glycerol. J Cell Physiol. 1981 Sep;108(3):439–443. doi: 10.1002/jcp.1041080318. [DOI] [PubMed] [Google Scholar]
  19. Lin T. Y., Timasheff S. N. Why do some organisms use a urea-methylamine mixture as osmolyte? Thermodynamic compensation of urea and trimethylamine N-oxide interactions with protein. Biochemistry. 1994 Oct 25;33(42):12695–12701. doi: 10.1021/bi00208a021. [DOI] [PubMed] [Google Scholar]
  20. Maroney A. C., Qureshi S. A., Foster D. A., Brugge J. S. Cloning and characterization of a thermolabile v-src gene for use in reversible transformation of mammalian cells. Oncogene. 1992 Jun;7(6):1207–1214. [PubMed] [Google Scholar]
  21. Martinez J., Georgoff I., Martinez J., Levine A. J. Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein. Genes Dev. 1991 Feb;5(2):151–159. doi: 10.1101/gad.5.2.151. [DOI] [PubMed] [Google Scholar]
  22. Michalovitz D., Halevy O., Oren M. Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53. Cell. 1990 Aug 24;62(4):671–680. doi: 10.1016/0092-8674(90)90113-s. [DOI] [PubMed] [Google Scholar]
  23. Pind S., Riordan J. R., Williams D. B. Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J Biol Chem. 1994 Apr 29;269(17):12784–12788. [PubMed] [Google Scholar]
  24. Santoro M. M., Liu Y., Khan S. M., Hou L. X., Bolen D. W. Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochemistry. 1992 Jun 16;31(23):5278–5283. doi: 10.1021/bi00138a006. [DOI] [PubMed] [Google Scholar]
  25. Sato S., Ward C. L., Krouse M. E., Wine J. J., Kopito R. R. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J Biol Chem. 1996 Jan 12;271(2):635–638. doi: 10.1074/jbc.271.2.635. [DOI] [PubMed] [Google Scholar]
  26. Schein C. H. Solubility as a function of protein structure and solvent components. Biotechnology (N Y) 1990 Apr;8(4):308–317. doi: 10.1038/nbt0490-308. [DOI] [PubMed] [Google Scholar]
  27. Stephen A. G., Trausch-Azar J. S., Ciechanover A., Schwartz A. L. The ubiquitin-activating enzyme E1 is phosphorylated and localized to the nucleus in a cell cycle-dependent manner. J Biol Chem. 1996 Jun 28;271(26):15608–15614. doi: 10.1074/jbc.271.26.15608. [DOI] [PubMed] [Google Scholar]
  28. Sturtevant J. M., Yu M. H., Haase-Pettingell C., King J. Thermostability of temperature-sensitive folding mutants of the P22 tailspike protein. J Biol Chem. 1989 Jun 25;264(18):10693–10698. [PubMed] [Google Scholar]
  29. Tatzelt J., Prusiner S. B., Welch W. J. Chemical chaperones interfere with the formation of scrapie prion protein. EMBO J. 1996 Dec 2;15(23):6363–6373. [PMC free article] [PubMed] [Google Scholar]
  30. Thomas P. J., Qu B. H., Pedersen P. L. Defective protein folding as a basis of human disease. Trends Biochem Sci. 1995 Nov;20(11):456–459. doi: 10.1016/s0968-0004(00)89100-8. [DOI] [PubMed] [Google Scholar]
  31. Welch W. J., Brown C. R. Influence of molecular and chemical chaperones on protein folding. Cell Stress Chaperones. 1996 Jun;1(2):109–115. doi: 10.1379/1466-1268(1996)001<0109:iomacc>2.3.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yancey P. H., Clark M. E., Hand S. C., Bowlus R. D., Somero G. N. Living with water stress: evolution of osmolyte systems. Science. 1982 Sep 24;217(4566):1214–1222. doi: 10.1126/science.7112124. [DOI] [PubMed] [Google Scholar]
  33. Yang Y., Janich S., Cohn J. A., Wilson J. M. 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 U S A. 1993 Oct 15;90(20):9480–9484. doi: 10.1073/pnas.90.20.9480. [DOI] [PMC free article] [PubMed] [Google Scholar]

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