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The Skaggs Institute
for Chemical Biology

Scientific Report 2008

Understanding and Ameliorating Protein-Misfolding Diseases

J.W. Kelly, S. Choi, E. Culyba, M.T.A. Dendle, D. Du, C. Fearns, A.A. Fuller, T.-W. Mu, A. Murray, D. Ong, J. Paulsson, E.T. Powers, P. Rao, M. Saure, R. Simkovsky, S. Siegel, J. Solomon, K. Usui, Y. Wang, I. Yonemoto, Z. Yu

Our main goal is to gain insight into the mechanisms of proteome maintenance that can be used to develop new therapeutic strategies to ameliorate protein-misfolding diseases when deficiencies in protein maintenance occur. Maintenance of the proteome (proteostasis) both inside and outside human cells is essential for development, reproduction, and successful aging. Deficiencies in proteostasis lead to many metabolic, oncologic, neurodegenerative, and cardiovascular disorders. We focus mostly on neurodegenerative diseases, and we benefit greatly from collaborations with J. Buxbaum, J.R. Yates, and W.E. Balch at Scripps Research and with A. Dillin at the Salk Institute for Biological Studies, La Jolla, California.

A Model for Protein Export from the Endoplasmic Reticulum

About one-third of all eukaryotic proteins, including all membrane proteins and secreted proteins, are folded in and then exported from the endoplasmic reticulum. Proteins are initially unstructured and must fold into well-defined structures to become functional. Unfortunately, proteins can also misfold, leaving them trapped in nonfunctional, sometimes aggregated, structures. Because of this inherent inefficiency in protein folding, the endoplasmic reticulum has a set of pathways that regulate protein folding and export. These pathways include an export pathway, which recognizes and exports properly folded proteins; an endoplasmic reticulum–associated degradation pathway, which recognizes and degrades unfolded or misfolded proteins; and a chaperoning pathway, which recognizes and recovers misfolded proteins. The efficiency with which a given protein is exported, defined as the rate of synthesis divided by the rate of export, depends on the interplay between the activities of these 3 pathways and the thermodynamics and kinetics of the folding and misfolding processes of the protein.

We recently described the FoldEx model of folding for export from the endoplasmic reticulum; this model was designed to semiquantitatively capture this interplay. In FoldEx, the activities of the export, degradation, and chaperone-mediated folding pathways are (most easily) controlled through the concentrations of the machineries that make up the pathways. The thermodynamics of folding are quantified by the equilibrium constant for folding, and the kinetics of folding or misfolding are quantified by the time required for folding or misfolding to reach its half-way point. The FoldEx model establishes that no single feature of protein folding energetics or endoplasmic reticulum biology dictates folding and transport efficiency. Instead, a network view provides insight into the basis for cellular diversity, disease origins, and protein homeostasis and predicts strategies for restoring protein homeostasis in protein-misfolding diseases.

Proteostasis Regulators and Pharmacologic Chaperones in Lysosomal Storage Diseases

Lysosomal storage diseases are loss-of-function diseases often caused by a mutation in one of the lysosomal enzymes. The mutation results in excessive misfolding and degradation of the enzyme within the endoplasmic reticulum instead of proper folding and trafficking of the enzyme to the lysosome. The resulting deficiency in lysosomal enzyme activity leads to accumulation of the substrate of the mutant lysosomal enzyme. At least 40 distinct lysosomal storage diseases have been identified; the most prevalent is Gaucher disease, which is caused by a deficiency in the activity of lysosomal glucocerebrosidase. Previously, we showed that in fibroblasts derived from the tissue of a patient with Gaucher disease, novel pharmacologic chaperones enhanced glucocerebrosidase activity up to 7.2-fold by binding directly to the enzyme and thereby stabilizing it.

More recently, we found that the innate proteostasis capacity of a cell can be enhanced with small molecules we call proteostasis regulators to fold mutated enzymes that would otherwise misfold and be degraded, resulting in increased trafficking of the mutated enzyme to the lysosome and increased function. We discovered that inhibiting L-type calcium channels with either diltiazem or verapamil partially restored enzyme homeostasis in 3 distinct lysosomal storage diseases: Gaucher disease, α-mannosidosis, and type IIIA mucopolysaccharidosis. The increased capacity of the endoplasmic reticulum to fold misfolding-prone proteins probably is due to a modest, calcium ion–mediated upregulation of a subset of molecular chaperones, including calnexin and calreticulin.

Correlating the Folding and Assembly Energetics of Transthyretin with Disease Phenotypes

Transthyretin, a tetrameric protein, is the primary transporter of retinol-binding protein and the secondary transporter of the thyroid hormone thyroxine. Transthyretin can dissociate, misfold, and aggregate, forming deposits that interfere with the normal functioning of several tissues or organs. Destabilized mutants of transthyretin are particularly prone to aggregation, but the precise energetic effects of the mutations are obscured by the linked folding and assembly equilibria of transthyretin. We used urea denaturation studies of transthyretin and several of its mutants to quantify the thermodynamically linked quaternary and tertiary structural stability to better understand the relationship between mutant folding energetics and amyloid disease phenotype.

Using a method of analysis that simultaneously accounts for the 2-step denaturation (tetramer dissociation followed by unfolding), we analyzed the stability of quaternary and tertiary structures of wild-type transthyretin and the V122I variant, which is linked to late-onset familial amyloid cardiomyopathy, the most common familial transthyretin amyloid disease. The results indicated that V122I transthyretin has a destabilized quaternary structure and a stable tertiary structure relative to wild-type transthyretin. We also examined 3 other variants of transthyretin: L55P, V30M, and A25T. We found that both the L55P mutant, associated with the most aggressive familial transthyretin amyloid disease, and the V30M mutant, the most common mutation associated with neuropathic forms of transthyretin amyloidosis, have complex denaturation pathways that cannot be fit to the 2-step denaturation model. Nevertheless, L55P transthyretin is clearly less stable than is wild-type transthyretin, primarily because the tertiary structure of L55P is unstable, although its quaternary structure is destabilized as well. Published data suggest that V30M transthyretin has stable quaternary structure but unstable tertiary structure. The A25T mutant, associated with CNS amyloidosis, is highly prone to aggregation and has drastically reduced quaternary and tertiary structural stability. The observed differences in stability among the disease-associated transthyretin variants highlight the complexity and the heterogeneity of transthyretin amyloid disease, an observation with important implications for the treatment of these diseases.

Aggregation of Amylin and its Processing Intermediates

Human amylin, or islet amyloid polypeptide, is a peptide cosecreted with insulin by the beta cells of the pancreatic islets of Langerhans. The 37-residue, C-terminally amidated human amylin peptide is derived from a proprotein that undergoes formation of disulfide bonds in the endoplasmic reticulum and then 4 enzymatic processing events in the immature secretory granule. Human amylin forms both intracellular and extracellular amyloid deposits in the pancreas of most patients with type 2 diabetes, likely reflecting compromised function of secretory cells. In addition, amylin-processing intermediates have been reported as components of intracellular amyloid in beta cells.

We investigated the amyloidogenicity of amylin and its processing intermediates in vitro. Under conditions mimicking those in immature secretory granules (37°C, pH 6), amylin forms amyloid aggregates more rapidly than its processing intermediates and its reduced counterparts form aggregates. Our results indicate that the amyloidogenicity of amylin and its processing intermediates is negatively correlated with net charge and charge at the C terminus. Although our conditions may not precisely reflect those of amyloidogenesis in vivo, the lower amyloidogenicity of the processing intermediates relative to amylin suggests that the presence of the intermediates in intracellular amyloid deposits in the increasingly stressed beta cells of patients with diabetes may be a consequence of general defects in protein homeostasis known to occur in diabetes rather than the result of the amylin processing intermediates acting as initiators of amyloid.


Balch, W.E., Morimoto, R.I., Dillin, A., Kelly, J.W. Adapting proteostasis for disease intervention. Science 319:916, 2008.

Bieschke, J., Siegel, S.J., Fu, J., Kelly, J.W. Alzheimer's Aβ peptides containing an isostructural backbone mutation afford distinct aggregate morphologies but analogous cytotoxicity: evidence for a common low-abundance toxic structure(s)? Biochemistry 47:50, 2008.

Gao, J., Kelly, J.W. Toward quantification of protein backbone-backbone hydrogen bonding energies: an energetic analysis of an amide-to-ester mutation in an α-helix within a protein. Protein Sci. 17:1096, 2008.

Hurshman Babbes, A.R., Powers, E.T., Kelly, J.W. Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its disease-associated variants: the relationship between stability and amyloidosis. Biochemistry 47:6969, 2008.

Johnson, S.M., Connelly, S., Wilson, I.A., Kelly, J.W. Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J. Med. Chem. 51:260, 2008.

Liu, F., Du, D., Fuller, A.A., Davoren, J.E., Wipf, P., Kelly, J.W., Gruebele, M. An experimental survey of the transition between two-state and downhill protein folding scenarios. Proc. Natl. Acad. Sci. U. S. A. 105:2369, 2008.

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.

Mu, T.-W., Ong, D.S.T., Wang, Y.-J., Balch, W.E., Yates, J.R. III, Segatori, L., Kelly, J.W. Chemical and biological approaches synergize to ameliorate protein-folding diseases. Cell 134:769, 2008.

Reixach, N., Foss, T.R., Santelli, E., Pascual, J., Kelly, J.W. Human-murine transthyretin heterotetramers are kinetically stable and non-amyloidogenic: a lesson in the generation of murine models of diseases involving oligomeric proteins. J. Biol. Chem. 283:2098, 2008.

Wiseman, R.L., Powers, E.T., Buxbaum, J.N., Kelly, J.W., Balch, W.E. An adaptable standard for protein export from the endoplasmic reticulum. Cell 131:809, 2007.

Yonemoto, I.T., Kroon, G.J.A., Dyson, H.J., Balch, W.E., Kelly, J.W. Amylin proprotein processing generates progressively more amyloidogenic peptides that initially sample the helical state. Biochemistry 47:9900, 2008.


Jeffery W. Kelly, Ph.D.
Lita Annenberg Hazen Professor of Chemistry

Dean, Graduate and Postgraduate Studies

Kelly Web Site