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Endoplasmic reticulum Ca2+ increases enhance mutant glucocerebrosidase proteostasis

An Erratum to this article was published on 01 August 2010

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Abstract

Altering intracellular calcium levels is known to partially restore mutant enzyme homeostasis in several lysosomal storage diseases, but why? We hypothesized that endoplasmic reticulum (ER) calcium increases enhance the folding, trafficking and function of these mutant misfolding- and degradation-prone lysosomal enzymes by increasing chaperone function. Here we report that increasing ER calcium levels by reducing ER calcium efflux through the ryanodine receptor, using antagonists or RNAi, or by promoting ER calcium influx by SERCA2b overexpression enhances mutant glucocerebrosidase (GC) homeostasis in cells derived from individuals with Gaucher's disease. Post-translational regulation of the calnexin folding pathway by an elevated ER calcium concentration seems to enhance the capacity of this chaperone system to fold mutant misfolding-prone enzymes, increasing the folded mutant GC population that can engage the trafficking receptor at the expense of ER-associated degradation, increasing the lysosomal GC concentration and activity.

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Figure 1: siRNA-mediated knockdown of RyR isoforms partially restores L444P GC proteostasis.
Figure 2: Ryanodine receptor antagonists act as GC proteostasis regulators.
Figure 3: Overexpression of the SERCA2b Ca2+ influx pump enhances mutant GC proteostasis.
Figure 4: Antagonists of the RyRs increase [Ca2+]ER in fibroblasts and HeLa cells, but do not activate the HSR and UPR in the L444P fibroblasts.
Figure 5: Overexpression of calnexin partially restores mutant GC proteostasis.
Figure 6: The RyR antagonist category GC proteostasis regulators post-translationally regulate the chaperone activity of calnexin in a Ca2+-dependent manner.

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Change history

  • 14 May 2010

    In the version of this article initially published online, three errors of sense were introduced into the text. Also, Figure 5b was incorrectly cited where Figure 5c should have been cited and an additional gene was erroneously listed as a chaperone not changing after diltiazem or dantrolene treatment in reference to Supplementary Figure 9a. These errors have been corrected for the print, PDF and HTML versions of this article.

  • 21 May 2010

    In the version of this article initially published, in the main text one of the cell lines used was erroneously given the mutant designation L444P in reference to Figure 6a and another cell line did not include this designation in reference to Supplementary Figure 11a,b. The errors have been corrected in the HTML and PDF versions of the article.

References

  1. Balch, W.E., Morimoto, R.I., Dillin, A. & Kelly, J.W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    Article  CAS  Google Scholar 

  2. Deuerling, E. & Bukau, B. Chaperone-assisted folding of newly synthesized proteins in the cytosol. Crit. Rev. Biochem. Mol. Biol. 39, 261–277 (2004).

    Article  CAS  Google Scholar 

  3. Tang, Y.C., Chang, H.C., Chakraborty, K., Hartl, F.U. & Hayer-Hartl, M. Essential role of the chaperonin folding compartment in vivo. EMBO J. 27, 1458–1468 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Bukau, B., Weissman, J. & Horwich, A. Molecular chaperones and protein quality control. Cell 125, 443–451 (2006).

    Article  CAS  Google Scholar 

  5. Morimoto, R.I. & Cuervo, A.M. Protein homeostasis and aging: taking care of proteins from the cradle to the grave. J. Gerontol. Ser. A Biol. Sci. Med. Sci. 64A, 167–170 (2009).

    Article  CAS  Google Scholar 

  6. Westerheide, S.D., Anckar, J., Stevens, S.M., Sistonen, L. & Morimoto, R.I. Stress-inducible regulation of heat shock factor 1 by the deacetylase SIRT1. Science 323, 1063–1066 (2009).

    Article  CAS  Google Scholar 

  7. Dai, C., Whitesell, L., Rogers, A.B. & Lindquist, S. Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell 130, 1005–1018 (2007).

    Article  CAS  Google Scholar 

  8. Ron, D. & Walter, P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell Biol. 8, 519–529 (2007).

    Article  CAS  Google Scholar 

  9. Schroder, M. & Kaufman, R.J. The mammalian unfolded protein response. Annu. Rev. Biochem. 74, 739–789 (2005).

    Article  Google Scholar 

  10. Gidalevitz, T., Ben-Zvi, A., Ho, K.H., Brignull, H.R. & Morimoto, R.I. Progressive disruption of cellular protein folding in models of polyglutamine diseases. Science 311, 1471–1474 (2006).

    Article  CAS  Google Scholar 

  11. Wang, X. et al. Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 127, 803–815 (2006).

    Article  CAS  Google Scholar 

  12. Mu, T.W., Fowler, D.M. & Kelly, J.W. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis. PLoS Biol. 6, e26 (2008).

    Article  Google Scholar 

  13. Mu, T.W. et al. Chemical and biological approaches synergize to ameliorate protein-folding diseases. Cell 134, 769–781 (2008).

    Article  CAS  Google Scholar 

  14. Cohen, E., Bieschke, J., Perciavalle, R.M., Kelly, J.W. & Dillin, A. Opposing activities protect against age-onset proteotoxicity. Science 313, 1604–1610 (2006).

    Article  CAS  Google Scholar 

  15. Morley, J.F., Brignull, H.R., Weyers, J.J. & Morimoto, R.I. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 99, 10417–10422 (2002).

    Article  Google Scholar 

  16. Futerman, A.H. & van Meer, G. The cell biology of lysosomal storage disorders. Nat. Rev. Mol. Cell Biol. 5, 554–565 (2004).

    Article  CAS  Google Scholar 

  17. Sawkar, A.R. et al. Chemical chaperones increase the cellular activity of N370S beta-glucosidase: a therapeutic strategy for Gaucher disease. Proc. Natl. Acad. Sci. USA 99, 15428–15433 (2002).

    Article  CAS  Google Scholar 

  18. Sawkar, A.R., D'Haeze, W. & Kelly, J.W. Therapeutic strategies to ameliorate lysosomal storage disorders—a focus on Gaucher disease. Cell. Mol. Life Sci. 63, 1179–1192 (2006).

    Article  CAS  Google Scholar 

  19. Schmitz, M., Alfalah, M., Aerts, J., Naim, H.Y. & Zimmer, K.P. Impaired trafficking of mutants of lysosomal glucocerebrosidase in Gaucher's disease. Int. J. Biochem. Cell Biol. 37, 2310–2320 (2005).

    Article  CAS  Google Scholar 

  20. Ron, I. & Horowitz, M. ER retention and degradation as the molecular basis underlying Gaucher disease heterogeneity. Hum. Mol. Genet. 14, 2387–2398 (2005).

    Article  CAS  Google Scholar 

  21. Schueler, U.H. et al. Correlation between enzyme activity and substrate storage in a cell culture model system for Gaucher disease. J. Inherit. Metab. Dis. 27, 649–658 (2004).

    Article  CAS  Google Scholar 

  22. Zimmer, K.-P. et al. Intracellular transport of acid β-glucosidase and lysosome-associated membrane proteins is affected in Gaucher's disease (G202R mutation). J. Pathol. 188, 407–414 (1999).

    Article  CAS  Google Scholar 

  23. Sawkar, A.R. et al. Chemical chaperones and permissive temperatures alter the cellular localization of Gaucher disease associated glucocerebrosidase variants. ACS Chem. Biol. 1, 235–251 (2006).

    Article  CAS  Google Scholar 

  24. Desnick, R.J. & Schuchman, E.H. Enzyme replacement and enhancement therapies: Lessons from lysosomal disorders. Nat. Rev. Genet. 3, 954–966 (2002).

    Article  CAS  Google Scholar 

  25. Ohashi, T. et al. Characterization of human glucocerebrosidase from different mutant alleles. J. Biol. Chem. 266, 3661–3667 (1991).

    CAS  PubMed  Google Scholar 

  26. Korkotian, E. et al. Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons. J. Biol. Chem. 274, 21673–21678 (1999).

    Article  CAS  Google Scholar 

  27. Lloyd-Evans, E. et al. Glucosylceramide and glucosylsphingosine modulate calcium mobilization from brain microsomes via different mechanisms. J. Biol. Chem. 278, 23594–23599 (2003).

    Article  CAS  Google Scholar 

  28. Pelled, D. et al. Enhanced calcium release in the acute neuronopathic form of Gaucher disease. Neurobiol. Dis. 18, 83–88 (2005).

    Article  CAS  Google Scholar 

  29. Valdivia, H.H., Valdivia, C., Ma, J.J. & Coronado, R. Direct binding of verapamil to the ryanodine receptor channel of sarcoplasmic-reticulum. Biophys. J. 58, 471–481 (1990).

    Article  CAS  Google Scholar 

  30. Shoshan-Barmatz, V., Pressley, T.A., Higham, S. & Kraus-Friedmann, N. Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic-reticulum—differences between liver and skeletal-muscle. Biochem. J. 276, 41–46 (1991).

    Article  CAS  Google Scholar 

  31. Zalk, R., Lehnart, S.E. & Marks, A.R. Modulation of the ryanodine receptor and intracellular calcium. Annu. Rev. Biochem. 76, 367–385 (2007).

    Article  CAS  Google Scholar 

  32. Ward, A., Chaffman, M.O. & Sorkin, E.M. Dantrolene—a review of its pharmacodynamic and pharmacokinetic properties and therapeutic use in malignant hyperthermia, the neuroleptic malignant syndrome and an update of its use in muscle spasticity. Drugs 32, 130–168 (1986).

    Article  CAS  Google Scholar 

  33. Kang, J.J., Hsu, K.S. & Linshiau, S.Y. Effects of bipyridylium compounds on calcium-release from triadic vesicles isolated from rabbit skeletal-muscle. Br. J. Pharmacol. 112, 1216–1222 (1994).

    Article  CAS  Google Scholar 

  34. Berridge, M.J. Inositol trisphosphate and calcium signaling. Nature 361, 315–325 (1993).

    Article  CAS  Google Scholar 

  35. Vangheluwe, P., Raeymaekers, L., Dode, L. & Wuytack, F. Modulating sarco(endo)plasmic reticulum Ca2+ ATPase 2 (SERCA2) activity: Cell biological implications. Cell Calcium 38, 291–302 (2005).

    Article  CAS  Google Scholar 

  36. Palmer, A.E., Jin, C., Reed, J.C. & Tsien, R.Y. Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc. Natl. Acad. Sci. USA 101, 17404–17409 (2004).

    Article  CAS  Google Scholar 

  37. Michalak, M., Parker, J.M.R. & Opas, M. Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum. Cell Calcium 32, 269–278 (2002).

    Article  CAS  Google Scholar 

  38. Schrag, J.D. et al. The structure of calnexin, an ER chaperone involved in quality control of protein folding. Mol. Cell 8, 633–644 (2001).

    Article  CAS  Google Scholar 

  39. David, V., Hochstenbach, F., Rajagopalan, S. & Brenner, M.B. Interaction with newly synthesized and retained proteins in the endoplasmic-reticulum suggests a chaperone function for human integral membrane-protein IP90 (calnexin). J. Biol. Chem. 268, 9585–9592 (1993).

    CAS  PubMed  Google Scholar 

  40. Lamb, H.K. et al. The affinity of a major Ca2+ binding site on GRP78 is differentially enhanced by ADP and ATP. J. Biol. Chem. 281, 8796–8805 (2006).

    Article  CAS  Google Scholar 

  41. Apperizeller-Herzog, C. & Ellgaard, L. The human PDI family: Versatility packed into a single fold. Biochim. Biophys. Acta-Mol. Cell Res. 1783, 535–548 (2008).

    Article  Google Scholar 

  42. Wilson, C.M., Farmery, M.R. & Bulleid, N.J. Pivotal role of calnexin and mannose trimming in regulating the endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain. J. Biol. Chem. 275, 21224–21232 (2000).

    Article  CAS  Google Scholar 

  43. Keller, S.H., Lindstrom, J. & Taylor, P. Inhibition of glucose trimming with castanospermine reduces calnexin association and promotes proteasome degradation of the alpha-subunit of the nicotinic acetylcholine receptor. J. Biol. Chem. 273, 17064–17072 (1998).

    Article  CAS  Google Scholar 

  44. Wang, J. & White, A.L. Role of calnexin, calreticulin, and endoplasmic reticulum mannosidase I in apolipoprotein(a) intracellular targeting. Biochemistry 39, 8993–9000 (2000).

    Article  CAS  Google Scholar 

  45. Brockmeier, A. & Williams, D.B. Potent lectin-independent chaperone function of calnexin under conditions prevalent within the lumen of the endoplasmic reticulum. Biochemistry 45, 12906–12916 (2006).

    Article  CAS  Google Scholar 

  46. Thammavongsa, V., Mancino, L. & Raghavan, M. Polypeptide substrate recognition by calnexin requires specific conformations of the calnexin protein. J. Biol. Chem. 280, 33497–33505 (2005).

    Article  CAS  Google Scholar 

  47. Tjoelker, L.W. et al. Human, mouse and rat calreticulin cDNA cloning—identification of potential calcium-binding motifs and gene localization to human-chromosome-5. Biochemistry 33, 3229–3236 (1994).

    Article  CAS  Google Scholar 

  48. Corbett, E.F. et al. The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment. J. Biol. Chem. 275, 27177–27185 (2000).

    CAS  PubMed  Google Scholar 

  49. Powers, E.T., Morimoto, R.I., Dillin, A., Kelly, J.W. & Balch, W.E. Biological and chemical approaches to diseases of proteostasis deficiency. Annu. Rev. Biochem. 78, 23.1–33 (2009).

    Article  Google Scholar 

  50. Le, A., Steiner, J.L., Ferrell, G.A., Shaker, J.C. & Sifers, R.N. Association between calnexin and a secretion-incompetent variant of human α1-antitrypsin. J. Biol. Chem. 269, 7514–7519 (1994).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank the following for their generosity in providing us with plasmids: W.E. Balch (The Scripps Research Institute) for pcDNA3.1+VSVG, E. Beutler (The Scripps Research Institute) for WT GC cDNA, M. Brenner (Harvard Medical School) for Apr-M8-CNX, K. Green (University of California, Irvine) for SERCA2 cDNA and T. Mizushima (Kumamoto University) for pCR(HA) and pcDNA3.1-GRP78. We thank H. Aerts (University of Amsterdam) for the mouse monoclonal anti-GC 8E4, W.E. Balch (The Scripps Research Institute) for mouse monoclonal anti-VSVG, M. Fukuda (Burnham Institute) for the rabbit anti-LAMP2 and C. Fearns for critical feedback on the manuscript. This work was supported by the NIH (DK75295), the Skaggs Institute for Chemical Biology and the Lita Annenberg Hazen Foundation. A.E.P. was supported by the NIH (GM084027).

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Authors

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D.S.T.O. performed the majority of the experiments, analyzed the data and wrote the initial draft of the paper. T.-W.M. performed the initial RyR inhibitor experiments and all the Ca2+ measurement experiments and collaborated on the analysis of the data. The ER Ca2+ levels were measured in the A.E.P. laboratory. J.W.K. supervised the work and managed the final publication.

Corresponding author

Correspondence to Jeffery W Kelly.

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Competing interests

J.W.K. is a cofounder, shareholder and paid consultant for Proteostasis Therapeutics, Inc., and although this company is not now pursuing any lysosomal storage diseases, that could happen in the future.

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Supplementary Figures 1–13, Supplementary Table 1 and Supplementary Methods (PDF 593 kb)

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Ong, D., Mu, TW., Palmer, A. et al. Endoplasmic reticulum Ca2+ increases enhance mutant glucocerebrosidase proteostasis. Nat Chem Biol 6, 424–432 (2010). https://doi.org/10.1038/nchembio.368

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