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The Skaggs Institute
for Chemical Biology

Understanding the Molecular Mechanisms of Protein Misfolding Diseases

J.W. Kelly, L. Bazhenova, J. Bieschke, D. Bosco, P. Braun, M.T.A. Dendle, W. D’Haeze, T. Foss, D. Fowler, K. Frankenfield, Y. Fu, J. Gao, M.-Y. Gao, A. Hurshman, S. Johnson, E.T. Powers, A. Sawkar, S. Siegel, J.-Y. Suk, L. Wiseman, I. Yonemoto, Z. Yu, Q. Zhang

Our goal is to understand molecular mechanisms of protein folding and of protein misfolding, which leads to neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. We use cell biological approaches, in collaboration with W.E. Balch, Scripps Research, spectroscopic and biophysical approaches in combination with chemical synthesis, and animal studies.

Transthyretin Amyloidogenesis

As a result of a mutation and/or denaturation stress associated with aging and/or oxidative stress, the transthyretin tetramer dissociates, and subsequent changes in the tertiary structure of the monomer make the monomer competent to misassemble into aggregates, including amyloid fibrils associated with numerous transthyretin amyloid diseases. We synthesized various structurally distinct small molecules that stabilize the native tetramer, thereby inhibiting the rate-limiting dissociation required for amyloidogenesis.

For example, bisarylaldoxime ethers substituted with a carboxylic acid on one aromatic ring and halogens or a trifluoromethyl group on the other aryl ring substantially inhibit the formation of transthyretin fibrils, as do oxazoles with a carboxyl group at C-4, a 3,5-dichlorophenyl group at C-2, and an ethyl, a propyl, or a trifluoromethyl group at C-5. Such small molecules might be suitable candidates for drugs for transthyretin amyloidoses, as illustrated by diflunisal, which is now being tested in humans in a multicenter placebo-controlled clinical trial.

We also showed that only the most destabilized transthyretin variants are degraded in the endoplasmic reticulum, and then only in certain tissues. We discovered that endoplasmic reticulum–assisted folding determines protein secretion in a tissue-specific manner, and we propose that its competition with endoplasmic reticulum–associated degradation may explain the appearance of tissue-selective amyloid diseases, especially for highly destabilized transthyretin variants that lead to CNS-selective disease, because the CNS is the only tissue that can secrete these highly unstable transthyretin variants.

We also investigated the role of the amino acid Cys10 in transthyretin amyloidogenicity. This amino acid is present in each transthyretin monomer and may occur in a mixed disulfide form. The Cys10 mixed disulfide form of Val30Met, an important disease-associated variant, had diminished protein stability and enhanced amyloidogenicity. However, in contrast to Val30Met, the double mutant Cys10Ser/Val30Met, which cannot make mixed disulfides, is nonamyloidogenic in transgenic mice, suggesting that the formation of mixed disulfides influences transthyretin amyloidosis in vivo.

Oxidative Metabolites and Protein Aggregation

In collaboration with P. Wentworth and R.A. Lerner, Scripps Research, we discovered that oxidative metabolites of cholesterol can covalently modify amyloid β-peptides (Aβ), dramatically accelerating the amyloidogenesis of these peptides, which are associated with Alzheimer’s disease. Metabolic modification of Aβ amyloidogenesis occurs via a 2-step mechanism involving the energetically downhill assembly of spherical aggregates by Aβ-metabolite adducts before the generation of fibrillar aggregates. This mechanism may explain the formation of Aβ aggregates in the brain at very low concentrations.

α-Synucleinopathies, including Parkinson’s disease and dementia with Lewy bodies, are characterized by cytoplasmic α-synuclein–rich aggregates within degenerating dopaminergic neurons in the substantia nigra. Clinical observations suggest a correlation between oxidative stress or inflammation and Parkinson’s disease. We are exploring the hypothesis that reactive oxygen species react with metabolites to generate oxidized metabolites that can interact with α-synuclein, triggering misfolding and subsequent aggregation. We aim to determine whether such metabolites accelerate the aggregation of α-synuclein in the same way they accelerate the aggregation of Aβ. Those studies will provide better insight into the correlation between oxidative stress and α-synucleinopathies.

β-Sheet Folding

We have studied the role of backbone hydrogen bonds in the folding kinetics and thermodynamics of the PIN WW domain, a 34-residue protein composed of 3 β-strands and 2 intervening loops. For these studies, we used amide-to-ester backbone mutations, alterations that perturb hydrogen bonding but do not affect backbone conformational preferences or the structure of side chains. Thermodynamic analysis indicated that the location of a backbone hydrogen bond determines the extent of protein destabilization caused by the elimination of a hydrogen bond by amide-to-ester mutation. Elimination of buried hydrogen bonds in the hydrophobic core substantially destabilizes the PIN WW domain; in contrast, elimination of hydrogen bonds present at or near loops exposed to solvent is only slightly destabilizing. In addition, we showed that the destabilization of the PIN WW domain was greater when a backbone hydrogen-bond donor was eliminated than when a hydrogen-bond acceptor was weakened. These findings are important because they suggest that only a subset of hydrogen bonds is energetically important in protein folding.

Aggregation of the Prion Protein

The N terminus of the recombinant human prion protein (PrP) is required for oligomerization of the protein in a neutral pH environment. We observed that the full-length PrP 23–231 forms a mixture of monomers, dimers, and trimers, whereas PrP 90–231, a truncated form lacking the N terminus, exists as a monomer. The later steps along the aggregation pathway are considerably different for these 2 prion protein structures. In the first step, PrP 23–231 forms a few large aggregates via a nucleated polymerization in which the nucleus is trimeric. The second step starts upon monomer depletion and is characterized by the formation of large aggregates that coincides with an increased turbidity of the assay solutions. The formation of the large aggregates is probably due to the association of existing aggregates.

The initial stages of aggregation are similar for PrP 90–231 and PrP 23–231; however, monomer depletion occurs much more slowly for PrP 90–231. We also observed that if a second stage of aggregation occurs, it must occur in a very inefficient way. The aggregates formed by PrP 90–231 are much smaller than those formed by the full-length prion protein, and direct association does not seem to take place. These findings are important for determining the optimal sequence to be used for screening for small molecules that prevent prion diseases.

Familial Amyloidosis of Finnish Type

Familial amyloidosis of Finnish type is caused by the D187N/Y mutation in plasma gelsolin. This disease is characterized by amyloid deposits composed of 5- and 8-kD internal fragments of plasma gelsolin. The loss of a calcium-binding site in domain 2 due to the D187N/Y mutation allows aberrant cleavage of furin in the Golgi apparatus, yielding a 68-kD fragment. This fragment is then cleaved by a transmembrane matrix metalloprotease, resulting in 5- and 8-kD fragments that are deposited as amyloid fibrils in the extracellular matrix. Sulfonated glycosaminoglycans accelerate gelsolin amyloidogenesis in vitro, possibly explaining the tissue selectivity of these diseases. Using a mouse model of familial amyloidosis of Finnish type, we are investigating treatment of the disease with inhibitors of furin, inhibitors of the matrix metalloprotease, and glycosaminoglycan antagonists.

Chemical Chaperones

Chemical chaperones are small molecules that bind and stabilize misfolded proteins in the endoplasmic reticulum and reduce the tendency of the proteins to be degraded by the proteasome. The N370S mutation in the lysosomal hydrolase glucocerebrosidase leads to degradation of the protein instead of folding and trafficking, and the subsequent accumulation of glucosylceramide in the lysosomes leads to Gaucher disease, the most common lysosomal storage disorder. We have synthesized small molecules, including N-(n-nonyl)deoxynojirimycin, that increase the activity of N370S glucocerebrosidase in a cell line derived from a patient with Gaucher disease. Binding of the small molecules stabilizes the glucocerebrosidase variant in the endoplasmic reticulum, allowing the enzyme to be trafficked successfully from the endoplasmic reticulum to the lysosome.

Recently, we assessed the amenability of other disease-associated glucocerebrosidase mutants, including G202R and L444P, to chemical chaperoning, and we are seeking new classes of small-molecule chaperones, such as deoxynojirimycin analogs and (iminosugar) C-glycosides. We found that other glucocerebrosidase mutants are amenable to chemical chaperoning, and a subset of the compounds tested increased the activity of multiple mutants. Interestingly, the recent elucidation of the x-ray crystal structures of native glucocerebrosidase and glucocerebrosidase bound to an irreversible inhibitor provides additional insight in the design of chemical chaperones.


Bieschke, J., Zhang, Q., Powers, E.T., Lerner, R.A., Kelly, J.W. Oxidative metabolites accelerate Alzheimer’s amyloidogenesis by a two-step mechanism, eliminating the requirement for nucleation. Biochemistry 44:4977, 2005.

Deechongkit, S., Dawson, P.E., Kelly, J.W. Toward assessing the position-dependent contributions of backbone hydrogen bonding to β-sheet folding thermodynamics employing amide-to-ester perturbations. J. Am. Chem. Soc. 126:16762, 2004.

Deechongkit, S., Powers, E.T., You, S.-L., Kelly, J.W. Controlling the morphology of cross β-sheet assemblies by rational design. J. Am. Chem. Soc. 127:8562, 2005.

Foss, T.R., Kelker, M.S., Wiseman, R.L., Wilson, I.A., Kelly, J.W. Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis. J. Mol. Biol. 347:841, 2005.

Frankenfield, K.N., Powers, E.T., Kelly, J.W. Influence of the N-terminal domain on the aggregation properties of the prion protein. Protein Sci. 14:2154, 2005.

Johnson, S.M., Petrassi, H.M., Palaninathan, S.K., Mohamedmohaideen, N.N., Purkey, H.E., Nichols, C., Chiang, K.P., Walkup, T., Sacchettini, J.C., Sharpless, K.B., Kelly, J.W. Bisaryloxime ethers as potent inhibitors of transthyretin amyloid fibril formation. J. Med. Chem. 48:1576, 2005.

Nguyen, H., Jäger, M., Kelly, J.W., Gruebele, M. Engineering a β-sheet protein toward the folding speed limit. J. Phys. Chem. B 109:15182, 2005.

Petrassi, H.M., Johnson, S.M., Purkey, H.E., Chiang, K.P., Walkup, T., Jiang, X., Powers, E.T., Kelly, J.W. Potent and selective structure-based dibenzofuran inhibitors of transthyretin amyloidogenesis: kinetic stabilization of the native state. J. Am. Chem. Soc. 127:6662, 2005.

Premkumar, L., Sawkar, A.R., Boldin-Adamsky, S., Toker, L., Silman, I., Kelly, J.W., Futerman, A.H., Sussman, J.L. X-ray structure of human acid-β-glucosidase covalently bound to conduritol-B-epoxide: implications for Gaucher disease. J. Biol. Chem. 280:23815, 2005.

Purkey, H.E., Palaninathan, S.K., Kent, K.C., Smith, C., Safe, S.H., Sacchettini, J.C., Kelly, J.W. Hydroxylated polychlorinated biphenyls selectively bind transthyretin in blood and inhibit amyloidogenesis: rationalizing rodent PCB toxicity. Chem. Biol. 11:1719, 2004.

Razavi, H., Powers, E.T., Purkey, H.E., Adamski-Werner, S.L., Chiang, K.P., Dendle, M.T.A., Kelly, J.W. Design, synthesis, and evaluation of oxazole transthyretin amyloidogenesis inhibitors. Bioorg. Med. Chem. Lett. 15:1075, 2005.

Sekijima, Y., Wiseman, R.L., Matteson, J., Hammarström, P., Miller, S.R., Sawkar, A.R., Balch, W.E., Kelly, J.W. The biological and chemical basis for tissue-selective amyloid disease. Cell 121:73, 2005.

Wiseman, R.L., Green, N.S., Kelly, J.W. Kinetic stabilization of an oligomeric protein under physiological conditions demonstrated by a lack of subunit exchange: implications for transthyretin amyloidosis. Biochemistry 44:9265, 2005.

Wiseman, R.L., Johnson, S.M., Kelker, M.S., Foss, T., Wilson, I.A., Kelly, J.W. Kinetic stabilization of an oligomeric protein by a single ligand binding event. J. Am. Chem. Soc. 127:5540, 2005.

You, S.-L., Kelly, J.W. The total synthesis of bistratamides F-I. Tetrahedron 61:241, 2005.

You, S.-L., Kelly, J.W. Total synthesis of didmolamides A and B. Tetrahedron Lett. 46:2567, 2005.

Zhang, Q., Kelly, J.W. Cys-10 mixed disulfide modifications exacerbate transthyretin familial variant amyloidogenicity: a likely explanation for variable clinical expression of amyloidosis and the lack of pathology in C10S/V30M transgenic mice? Biochemistry 44:9079, 2005.


Jeffery W. Kelly, Ph.D.
Vice President, Academic Affairs, Scripps Research
Dean, Kellogg School of Science and Technology
Lita Annenberg Hazen Professor of Chemistry

Kelly Web Site