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Reclamation of Proteins from the Cellular Scrap Heap Jason E. Gestwicki*
Department of Pathology and the Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216
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ewly synthesized polypeptides that are unable to fold in a timely manner are targeted to the proteasome. While this quality control checkpoint is intended to avoid harmful accumulation of misfolded intermediates, premature disposal can also cause severe loss-offunction defects. In these cases, the levels of folded protein can sometimes be restored by treatment with that protein’s ligands. Because these compounds mimic the productive folding activity of the cell’s own chaperones, they are termed chemical chaperones. However, the mechanism used by these molecules to rescue otherwise doomed polypeptides has been uncertain. On page 235 of this issue, Sawkar et al. (1) provide our best glimpse of chemical chaperones in action. Using unstable glucocerebrosidase (GC) mutants as a model, Sawkar et al. show that inhibitors leverage their binding energy to favor productive folding in the environment of the endoplasmic reticulum. This allows the enzyme to escape capture by the quality control machinery. These important insights should facilitate the design of small molecules that snatch specific proteins from the cellular scrap heap. However, this nontraditional drug strategy requires a shift in thinking for chemists and biologists alike; counterintuitive concepts, such as active site inhibitors that essentially behave as agonists at the cellular level, will need to be mastered. The more we understand about the activity of chemical chaperones, the greater potential
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they have to treat the growing number of disorders recognized, broadly, as misfolding diseases. Most newly synthesized proteins require the assistance of cellular chaperones to interpret the folding information contained in their primary sequence. For example, peptides expressed into the lumen of the endoplasmic reticulum (ER) are subject to a large family of heat shock proteins, cochaperones, lectins, membrane-spanning translocons, and ubiquitin-conjugating enzymes (for recent reviews see refs 2 and 3). If prolonged failures are detected, this machinery also mediates the ER-associated degradation (ERAD) that eliminates the offending polypeptide. The choice between continued folding and terminal disposal is central to protein homeostasis and critical to the ongoing health of the cell: when misfolded intermediates are left unattended, they can disrupt neighboring proteins and nucleate cytotoxic aggregates. Mutations that leave a protein susceptible to misfolding are associated with numerous diseases (4, 5), including the neurodegenerative disorders (e.g., Alzheimer’s and Parkinson’s diseases), peripheral amyloidoses, cystic fibrosis, and certain lysosomal storage disorders (e.g., Gaucher disease, see below). Some of these diseases, such as Alzheimer’s, are characterized by the formation of proteaseresistant aggregates and an associated gain-of-function toxicity. Others are caused by loss-of-function defects; pathology arises
A B S T R A C T A growing number of diseases have been associated with protein misfolding. Thus, strategies that use small molecules to adjust folding tendencies have therapeutic potential. However, progress in this area has been hampered by an insufficient description of the molecular underpinnings of protein instability within the cell. In a recent report, a chemical approach was taken to probe the mechanism by which Gaucher disease associated mutations in glucocerebrosidase destabilize that enzyme and lead to its destruction. These studies provide a blueprint for the design of “chemical chaperones” for the exploration of cellular protein homeostasis and the treatment of misfolding diseases.
*To whom correspondence may be addressed. E-mail:
[email protected].
Published online May 19, 2006 10.1021/cb001784 CCC: $33.50 © 2006 by American Chemical Society
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Figure 1. Model for chemical chaperone-assisted stabilization of glucocerebrosidase. Mutants harboring destabilizing substitutions (red) fail to fold properly in the neutral ER lumen. These polypeptides are destined for removal by the proteasome. However, binding to chemical chaperones rescues these mutants and permits trafficking to the lysosome. After arrival in this compartment, the chemical chaperone is no longer required for folding and its dissociation restores enzymatic activity.
from the diminished activity of an essential protein. In perhaps the best-known example of this type, reduced cell surface levels of the cystic fibrosis transmembrane conductance regulator (CFTR) underlie pathology of that disease (6). Gaucher disease is one of approximately 40 lysosomal storage disorders in which defects arise from incomplete catabolism of glycosphingolipids (7). In Gaucher patients, harmful accumulation of glucosylceramide is caused by loss-of-function mutations in a specific lysosomal GC. While some of these mutants appear to encode enzymatically inactive products, other common mutations may simply destabilize the enzyme and mark it for ERAD. Because these later defects are distal from the active site, they do not directly impinge on enzymatic function. This suggests that, if the proteins were safeguarded through the ER’s quality control machinery, they would retain a high degree of enzymatic function. Consistent with this hypothesis, osmolytes that generally favor protein folding, such as dimethyl 202
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sulfoxide (DMSO) and glycerol, have been found to rescue lysosomal GC activity (8). Chemical chaperones were developed as a pharmacologically superior alternative to osmolytes (for recent reviews see refs 9–12). These small molecules are designed to specifically interact with enzymes and protect against misfolding. For example, in 1999, a competitive inhibitor of ␣-galactosidase was found to enhance total enzyme activity in cells harboring an unstable mutant (13). However, proper use of these reagents poses an interesting conundrum: how can an inhibitor be used to enhance an enzyme’s function? Our understanding of this apparent contradiction would directly benefit from a deeper mechanistic understanding of how chemical chaperones work. For example, how much of the lost folding energy needs to be restored to avoid ERAD? In what subcellular compartment must chaperoning occur? A key step forward in our understanding of this process is reported by Jeffery Kelly’s laboratory (1). The Kelly laboratory has previGESTWICKI
ously established that nonyl deoxynojirimycin, a glucocerebrosidase inhibitor, can rescue lysosomal GC activity in a Gaucher disease model (14). Now, for the first time, the Kelly laboratory has quantified the energetics of an unstable GC variant, N370S. They found that N370S is thermally unstable at the neutral pH of the ER but more stable (and partially functional) in the lysosome’s acidic environment. Importantly, nonyl deoxynorjirimycin was able to partly restore folding energy at neutral pH. This result strongly supports a model (Figure 1) in which membrane-permeable ligands directly facilitate folding of nascent polypeptide in the ER. Following successful trafficking, drug is released and enzymatic function restored. In addition to these mechanistic insights, the Kelly laboratory also reports that drug treatment reverses trafficking defects and restores lysosomal enzymatic activity in fibroblasts derived from Gaucher disease patients expressing the N370S mutation. These physiologically significant findings affirm the potential therapeutic value of chemical chaperones. Finally, the mechanistic and cellular details gleaned from these studies provide the foundation for optimizing the activity of chemical chaperones. For example, next generation molecules might be designed to bind selectively at neutral pH with rapid dissociation in acidic compartments (e.g., using protonatable groups with an appropriate pKa). These compounds might be expected to have a superior therapeutic window, owing to their combination of high chaperone activity and low inhibitory potential. In addition to their promise as therapeutics for misfolding diseases, chemical chaperones might become important reagents for exploring the fundamental biology of protein homeostasis. Currently, inhibitors of cellular chaperones are among the best tools for these studies (15). These drugs rapidly inactivate the refolding machinery and reveal the biological consequences of www.acschemicalbiology.org
Point of
VIEW cellular chaperone function. For example, geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), was used to define this protein’s substantial role in p53 regulation (16). Chemical chaperones complement these reagents by enhancing the folding energy of specific targets. Used in combination, cellular chaperone inhibitors and chemical chaperones might illuminate how the decision is made to refold or degrade proteins. REFERENCES 1. Sawkar, A. R. (2006) ACS Chem. Biol. 1, 235–251. 2. Ahner, A., and Brodsky, J. L. (2004) Checkpoints in ER-associated degradation: excuse me, which way to the proteasome?, Trends Cell. Biol. 14, 474–478. 3. Meusser, B., Hirsch, C., Jarosch, E., and Sommer, T. (2005) ERAD: the long road to destruction, Nat. Cell. Biol. 7, 766–772. 4. Selkoe, D. (2003) Folding proteins in fatal ways, Nature 426, 900–904. 5. Stefani, M., and Dobson, C. M. (2003) Protein aggregation and aggregation toxicity: new insights into protein folding, misfolding diseases and biological evolution, J. Mol. Med. 81, 678–699. 6. Kopito, R. R. (1999) Biosynthesis and degradation of CFTR, Physiol. Rev. 79, S167–S173. 7. Sawkar, A. R., D’Haeze, W., and Kelly, J. W. (2006) Therapeutic strategies to ameliorate lysosomal storage disorders—a focus on Gaucher disease, Cell. Mol. Life Sci., published online Mar 29, http://dx.doi.org/10.1007/s00018-005-5437-0.
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8. Brown, C. R., HongBrown, L. Q., and Welch, W. J. (1997) Correcting temperature-sensitive protein folding defects, J. Clin. Invest. 99, 1432–1444. 9. Ulloa-Aguirre, A., Janovick, J. A., Brothers, S. P., and Conn, P. M. (2004) Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease, Traffic 5, 821–837. 10. Perlmutter, D. H. (2002) Chemical chaperones: a pharmacological strategy for disorders of protein folding and trafficking, Pediatr. Res. 52, 832–836. 11. Bernier, V., Lagace, M., Bichet, D. G., and Bouvier, M. (2004) Pharmacological chaperones: potential treatment for conformational diseases, Trends Endocrinol. Metab. 15, 222–228. 12. Cohen, F. E., and Kelly, J. W. (2003) Therapeutic approaches to protein misfolding diseases, Nature 426, 905–909. 13. Fan, J. Q., Ishii, S., Asano, N., and Suzuki, Y. (1999) Accelerated transport and a maturation of lysosomal a-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor, Nat. Med. 5, 112–115. 14. Sawkar, A. R., Adamski-Werner, S. L., Cheng, W.-C., Wong, C.-H., Beutler, E., Zimmer, K.-P., and Kelly, J. W. (2005) Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles, Chem. Biol. 12, 1235–1244. 15. Smith, D. F., Whitesell, L., and Katsanis, E. (1998) Molecular chaperones: biology and prospects for pharmacological intervention, Pharm. Rev. 50, 493–513. 16. Galigniana, M. D., Harrell, J. M., O-Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004) Hsp90binding immunophilins link p53 to dynein during p53 transport to the nucleus, J. Biol. Chem. 279, 22483–22489.
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