Scientific Report 2006

Chemistry

Strategies to Ameliorate Protein Misfolding Diseases

J.W. Kelly, J. Bieschke, D.A. Bosco, E. Culyba, M.T.A. Dendle, W. D’Haeze, T.R. Foss, D.M. Fowler, Y. Fu, J. Gao, M.-Y. Gao, S.M. Johnson, T. Mu, E.T. Powers, A.R. Sawkar, L. Segatori, S. Siegel, J.Y. Suk, R.L. Wiseman, I. Yonemoto, Z. Yu

Our goal is to better understand the molecular mechanisms that lead to protein misfolding diseases, including Alzheimer’s, Parkinson’s, and Gaucher diseases, so that we can design new strategies to ameliorate such maladies. To accomplish this goal, we use organismal and cell biological disease models and spectroscopic and biophysical approaches in combination with chemical synthesis and molecular biology techniques. Successful collaborations with W.E. Balch, Department of Cell Biology, P. Wentworth, Jr., Department of Chemistry, and A. Dillin, the Salk Institute for Biological Studies, La Jolla, California, are critical to achieve our goals.

Transthyretin Amyloidogenesis

Transthyretin is a 55-kD homotetrameric protein that transports holo-retinol binding protein and L-thyroxine in the blood and cerebrospinal fluid. More than 99.5% of the transthyretin binding sites for L-thyroxine remain unoccupied in blood, and only about 50% of transthyretin tetramers in plasma are bound to a single holo-retinol binding protein. As a consequence of a mutation or denaturation stress associated with aging and/or oxidative stress, dissociation of the native transthyretin tetramer, the rate-limiting step for amyloidogenesis, followed by changes in the tertiary structure of the monomer make the monomeric subunits competent to misassemble into aggregates, including amyloid fibrils. The deposition of transthyretin amyloid is linked with a number of human diseases, including senile systemic amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy, and CNS-selective amyloidosis.

A suitable strategy to slow or prevent the formation of aggregates is to inhibit the rate-limiting dissociation of the transthyretin tetramer by making the native state more stable than the dissociative transition state. We have designed, synthesized, and characterized several classes of structurally distinct small molecules that bind to and stabilize the transthyretin tetramer. One of these molecules is being tested in phase 2/3 clinical trials for treatment of FAP. Compounds discovered in high-throughput screening tests, such as genistein, a natural compound present in soy products, also inhibit formation of fibrils of wild-type amyloid, as well as amyloidogenesis by the transthyretin variants V30M and V122I, 2 of the most common disease-associated variants. Furthermore, a clinical study in healthy human subjects indicated that kinetic stabilization of transthyretin mediated by orally administered diflunisal, a nonsteroidal anti-inflammatory agent also discovered by screening, should ameliorate transthyretin amyloidosis. The effect of diflunisal on FAP is currently being evaluated in a phase 3 clinical study.

Most likely the age-associated nature of neurodegenerative diseases such as the transthyretin amyloidoses can be explained by a shift from efficient to inefficient aggregate clearance, leading to increasing concentrations of the aggregates and proteotoxic effects. We showed that the reassembly of transthyretin homotetramers occurs via a monomer-dimer-trimer-tetramer pathway in which each step depends on the concentration of folded transthyretin monomers. This finding suggests that partitioning of transthyretin monomers between transthyretin tetramer reassembly and the aggregation pathway is correlated with the relative concentrations of reassembly intermediates and aggregates. The concentration of the aggregates presumably increases with aging because of inefficient aggregate clearance.

In another study, we found that subunits of the transthyretin variant R104H most likely act as an in vivo trans-suppressor of amyloidogenesis associated with the V30M variant. Compound heterozygotes with genes for the V30M and R104H variants did not show signs of the pathologic changes associated with FAP typical of heterozygotes with genes for the V30M variant and wild-type transthyretin; however, compound heterozygotes with genes for R104H and the aggressive mutation T59K on their second allele did have pathologic FAP effects similar to those of compound heterozygotes with genes for wild-type transthyretin and the T59K mutation.

We investigated the energetics of R104H homotetramers and mixed tetramers in a fashion analogous to that used to elucidate the mechanism of T119M transthyretin interallelic trans-suppression. We found that in contrast to T119M, R104H does not suppress aggregation by a kinetic stabilization mechanism. We showed that R104H may trans-suppress transthyretin aggregation by subtle thermodynamic stabilization of the transthyretin quaternary structure. This discovery suggests that R104H could protect compound heterozygotes from transthyretin aggregation in situations in which the mutation is mildly destabilizing. This finding supports the current clinical data associated with R104H in compound heterozygotes.

Aberrant Oxidative Metabolites Affect α-Synucleinopathies

The α-synucleinopathies are characterized by cytoplasmic α-synuclein-rich aggregates within degenerating dopaminergic neurons in the substantia nigra. Clinical observations suggest a correlation between oxidative stress/inflammation and protein misfolding diseases. We wished to determine whether oxidized metabolites accelerate the aggregation of α-synuclein, research that would shed light on the correlation between oxidative stress and sporadic α-synucleinopathies.

We found that overexpression of α-synuclein in a neuronal cell line is sufficient to increase the production of oxidative metabolites derived from the oxidation of cholesterol, presumably due to the production of reactive oxygen species. We showed that these oxidative metabolites are cytotoxic and that they significantly accelerate aggregation of α-synuclein in vitro. The experimental data suggest that the acceleration in aggregation occurs predominantly via a noncovalent mechanism. Overexpression of α-synuclein may lead to the production of reactive oxygen species, which stimulates the production of oxidative cholesterol metabolites that then accelerate α-synuclein aggregation. This acceleration may enhance local oxidative stress, resulting in a vicious cycle that eventually leads or contributes to α-synucleinopathies.

Although the exact role of α-synuclein in Lewy body disease and Parkinson’s disease remains to be elucidated, our findings add to an understanding of how aldehyde-based organic compounds formed as a result of aging and inflammation may contribute to neurodegenerative diseases.

Gelsolin Amyloidosis

Gelsolin amyloidogenesis occurs in persons who produce D187N/Y plasma gelsolin variants. This disease is characterized by amyloid deposits composed of 5- and 8-kD fragments of plasma gelsolin. The D187N/Y mutation abrogates calcium binding in domain 2, allowing aberrant furin cleavage in the Golgi apparatus during trafficking and yielding a 68-kD fragment. The fragment is then cleaved by the membrane type matrix metalloproteinase 1 (MT1-MMP), resulting in 5- and 8-kD fragments that are deposited as amyloid fibrils in the extracellular matrix. Fibroblasts from animals lacking the gene for MT1-MMP are incapable of generating 8- and 5-kD fragments from the 68-kD gelsolin fragment.

Biophysical studies indicated that gelsolin amyloid formation is substantially accelerated in the presence of the extracellular matrix component heparin. The extent of sulfation and the location and relative orientation of sulfate residues and the molecular weight are important factors in the heparin-mediated acceleration of gelsolin amyloidogenesis, possibly explaining the heavy deposition of gelsolin amyloid in the extracellular matrix. Most likely tissue-selective deposition of gelsolin amyloid is correlated with the localization of extracellular sulfated glycosaminoglycans, a notion supported by the colocalization of glycosaminoglycans with gelsolin amyloid in the extracellular space of gelsolin amyloidosis transgenic mice. These transgenic mice will be used to evaluate the effect of MT1-MMP inhibitors and glycosaminoglycan amyloid antagonists on gelsolin amyloidosis in vivo.

Chemical Chaperones and Gaucher Disease

Mutations in glucocerebrosidase, a lysosomal hydrolase, lead to an accumulation of glucosylceramide in the lysosome, causing Gaucher disease, the most common lysosomal storage disorder. Previously, we showed that N-(n-nonyl)deoxynojirimycin increases the activity of the glucocerebrosidase variant N370S in a cell line derived from tissue from a patient with Gaucher disease. This “chemical chaperoning” effect most likely is due to the binding of N-(n-nonyl)deoxynojirimycin to the native state of N370S, allowing the glucocerebrosidase to be trafficked from the endoplasmic reticulum to the lysosomes. Interestingly, the activity of G202R glucocerebrosidase, a variant retained in the endoplasmic reticulum, is also increased in the presence of chemical chaperones, suggesting that those chemical chaperones stimulated transport to the lysosomes.

Our results indicate that some chemical chaperones enhance the activity of distinct glucocerebrosidase variants to an extent thought to be sufficient to ameliorate Gaucher disease. Preliminary data suggest that certain glucocerebrosidase mutants most likely will need specifically designed chemical chaperones that target the compromised domain in order to facilitate proper trafficking and partial restoration of the function of the glucocerebrosidase.

Publications

Bosco, D.A., Fowler, D.M., Zhang, Q., Nieva, J., Powers, E.T., Wentworth, P., Jr., Lerner, R.A., Kelly, J.W. Elevated levels of oxidized cholesterol metabolites in Lewy body disease brains accelerate α-synuclein fibrilization [published correction appears in Nat. Chem. Biol. 2:346, 2006]. Nat. Chem. Biol. 2:249, 2006.

Deechongkit, S., Nguyen, H., Jäger, M., Powers, E.T., Gruebele, M., Kelly, J.W. β-Sheet folding mechanisms from perturbation energetics. Curr. Opin. Struct. Biol. 16:94, 2006.

Foss, T.R., Wiseman, R.L., Kelly, J.W. The pathway by which the tetrameric protein transthyretin dissociates. Biochemistry 44:15525, 2005.

Fowler, D.M., Koulov, A.V., Alory-Jost, C., Marks, M.S., Balch, W.E., Kelly, J.W. Functional amyloid formation within mammalian tissue. PLoS Biol. 4:e6, 2006.

Fu, Y., Bieschke, J., Kelly, J.W. E-Olefin dipeptide isostere incorporation into a polypeptide backbone enables hydrogen bond perturbation: probing the requirements for Alzheimer’s amyloidogenesis. J. Am. Chem. Soc. 127:15366, 2005.

Green, N.S., Foss, T.R., Kelly, J.W. Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis. Proc. Natl. Acad. Sci. U. S. A. 102:14545, 2005.

Johnson, S.M., Wiseman, R.L., Sekijima, Y., Green, N.S., Adamski-Werner, S.L., Kelly, J.W. Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a focus on the transthyretin amyloidoses. Acc. Chem. Res. 38:911, 2005.

Kelly, J.W., Balch, W.E. The integration of cell and chemical biology in protein folding. Nat. Chem. Biol. 2:224, 2006.

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

Page, L.J., Suk, J.Y., Huff, M.E., Lim, H.-J., Venable, J., Yates, J. III, Kelly, J.W., Balch, W.E. Metalloendoprotease cleavage triggers gelsolin amyloidogenesis. EMBO J. 24:4124, 2005.

Powers, E.T., Deechongkit, S., Kelly, J.W. Backbone-backbone H-bonds make context-dependent contributions to protein folding kinetics and thermodynamics: lessons from amide-to-ester mutations. Adv. Protein Chem. 72:39, 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.

Sawkar, A.R., Adamski-Werner, S.L., Cheng, W.-C., Wong, C.-H., Beutler, E., Zimmer, K.-P., Kelly, J.W. Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles. Chem. Biol. 12:1235, 2005.

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, 2006.

Sekijima, Y., Dendle, M.T., Wiseman, R.L., White, J.T., D’Haeze, W., Kelly, J.W. R104H may suppress transthyretin amyloidogenesis by thermodynamic stabilization, but not by the kinetic mechanism characterizing T119 trans-suppression. Amyloid 13:57, 2006.

Sörgjerd, K., Ghafouri, B., Jonsson, B.-H., Kelly, J.W., Blond, S.Y., Hammarström, P. Retention of misfolded mutant transthyretin by the chaperone BiP/GRP78 mitigates amyloidogensis. J. Mol. Biol. 356:469, 2006.

Suk, J.Y., Zhang, F., Balch, W.E., Linhardt, R.J., Kelly, J.W. Heparin accelerates gelsolin amyloidogenesis. Biochemistry 45:2234, 2006.

Wiseman, R.L., Powers, E.T., Kelly, J.W. Partitioning conformational intermediates between competing refolding and aggregation pathways: insights into transthyretin amyloid disease. Biochemistry 44:16612, 2005.