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Scientific Report 2004


Bioorganic and Biophysical Chemistry

J.W. Kelly, J. Bieschke, D. Bosco, P. Braun, S. Deechongkit, M.A. Dendle, T. Foss, D. Fowler, K. Frankenfield, Y. Fu, N. Green, M.E. Huff, A. Hurshman, S. Johnson, H.-J. Lim, E.T. Powers, A. Sawkar, Y. Sekijima, S. Siegel, J. Suk, S. Werner, I. Yonemoto, S. You, Z. Yu, Q. Zhang

Our goal is to improve our understanding of protein folding and misfolding and to determine how these processes are related to disease and how they can be manipulated with small molecules. We use biophysical methods in combination with chemical synthesis and protein engineering to accomplish these aims. Specific projects include the following.

Studies of Transthyretin Amyloid Diseases

Diseases of protein misfolding are becoming increasingly common in the United States as the population becomes older. Amyloid diseases, a major class of protein-misfolding diseases, are characterized by the systemic or tissue-localized deposition of a fibrillar, β-sheet–rich, protein aggregates known as amyloid. The fibrils associated with each amyloid disease are largely composed of a single protein, but this protein is different for each disease.

Transthyretin is a homotetrameric protein that is the primary carrier of the complex composed of vitamin A and the retinol-binding protein and the secondary carrier of thyroxine (the thyroid hormone) in plasma. Amyloid formation by transthyretin appears to cause several diseases, including senile systemic amyloidosis and familial amyloid polyneuropathy. Transthyretin amyloidogenesis begins with dissociation of the tetramer in response to a stress that causes denaturation (e.g., low pH, a hydrophobic environment). The resulting monomers can then partially denature and assemble into amyloid fibrils.

Using an engineered transthyretin monomer as a model system, we found that aggregation by transthyretin monomers occurs by a downhill polymerization mechanism; that is, every step along the amyloid formation pathway is favorable and fast relative to tetramer dissociation. Tetramer dissociation is therefore the rate-limiting step for transthyretin amyloid formation.

Because tetramer dissociation is the rate-limiting step, we proposed that it should be possible to treat transthyretin amyloid disease by stabilizing the transthyretin tetramer. This hypothesis is supported by the phenomenon of trans suppression, in which disease does not develop in heterozygotes who have both a disease-associated mutation and a trans suppressor mutation in their transthyretin. We showed that trans suppressor mutations inhibit amyloid formation by kinetically stabilizing (i.e., slowing the dissociation of) mixed transthyretin tetramers.

Similarly, we showed that binding of small molecules in the thyroxine-binding sites of transthyretin inhibits amyloid formation in vitro by stabilizing the native tetramer, thus increasing the energetic barrier for, and slowing, tetramer dissociation. We identified many small-molecule inhibitors of transthyretin amyloid formation, including diflunisal, a drug approved by the Food and Drug Administration. In collaboration with J. Buxbaum, Department of Molecular and Experimental Medicine, we are testing diflunisal and other compounds in mouse models and in human clinical trials as treatments for transthyretin amyloid disease.

In addition, in collaboration with W.E. Balch, Department of Cell Biology, we are using a combined biophysical and cell biological approach to understand the relationship between transthyretin mutations and the pathology of the diseases they cause. The transthyretin amyloid diseases with the earliest onsets and greatest severities are generally associated with mutations that strongly destabilize the transthyretin tetramer. However, the A25T and D18G mutants of transthyretin do not follow this trend. These mutants form extremely unstable tetramers (A25T) or do not form tetramers at all (D18G), but the onsets of their associated amyloid diseases do not occur until middle age. Furthermore, amyloid deposition of these mutants occurs in the CNS, which is unusual for transthyretin.

To explain this behavior, we hypothesized that the A25T and D18G transthyretins are intercepted by the quality control machinery of the protein secretion pathway in liver cells, where most of the transthyretin in the plasma is synthesized. The concentration of mutant transthyretin in plasma would therefore not be high enough to cause systemic amyloid disease. The transthyretin in the CNS, however, is produced by the choroid plexus, where the concentration of thyroxine is higher than it is in the liver. Thyroxine could bind to and stabilize the mutant transthyretin, allowing the mutant to bypass the quality control mechanism and to be secreted. The thyroxine would then dissociate, and the mutant transthyretin could misfold and form amyloid.

We were able to support our hypothesis with several observations and experiments. First, we found that the concentrations of the D18G and A25T transthyretins is low in the plasma of patients with these mutations. Second, we showed that the efficiency of secretion of transthyretin in cell lines correlated with a parameter that reflected both the thermodynamic and kinetic stability of a given transthyretin mutant (the “combined stability score”). The A25T and D18G transthyretin variants, in particular, had very low combined stability scores and were not efficiently secreted. In fact, we found that they underwent degradation associated with the endoplasmic reticulum. Finally, we showed that treating cells expressing A25T or D18G transthyretin with thyroxine (or other small molecules with an affinity for transthyretin) increased the efficiency of the secretion of the transthyretin.

Complementing Defects in Folding with Small-Molecule Chaperones

The N370S mutant of glucocerebrosidase, a lysosomal hydrolase, accumulates in the endoplasmic reticulum rather than being trafficked to the lysosome. This lack of trafficking causes the substrate of glucocerebrosidase to accumulate in the lysosome and leads to Gaucher disease, a lysosomal storage disease. Previously, we showed that the small molecule N-(n-nonyl)deoxynojirimicin can increase the activity of N370S glucocerebrosidase in a cell line derived from cells from a patient with Gaucher disease. An explanation for this increased activity is that the small molecule acts as a chemical chaperone for glucocerebrosidase; that is, binding of the small molecule could stabilize glucocerebrosidase and allow successful trafficking of the enzyme from the endoplasmic reticulum to the lysosome.

We recently expanded our studies of Gaucher disease to include more disease-associated glucocerebrosidase mutants and classes of small molecules. We found that several of the other glucocerebrosidase mutants are also amenable to chemical chaperoning, and several of the compounds tested could increase the activity of multiple mutants.


Adamski-Werner, S.L., Palaninathan, S.K., Sacchettini, J.C., Kelly, J.W. Diflunisal analogues stabilize the native state of transthyretin: potent inhibition of amyloidogenesis. J. Med. Chem. 47:355, 2004.

Cohen, F.E., Kelly, J.W. Therapeutic approaches to protein-misfolding diseases. Nature 426:905, 2003.

Deechongkit, S., Nguyen, H., Powers, E.T., Dawson, P.E., Gruebele, M., Kelly, J.W. Context dependent contributions of backbone hydrogen bonding to β-sheet folding energetics. Nature, in press.

Deechongkit, S., You, S.L., Kelly, J.W. Synthesis of all nineteen appropriately protected chiral α-hydroxy acid equivalents of the α-amino acids for Boc solid-phase depsi-peptide synthesis. Org. Lett. 6:497, 2004.

Green, N.S., Palaninathan, S.K., Sacchettini, J.C., Kelly, J.W. Synthesis and characterization of potent bivalent amyloidosis inhibitors that bind prior to transthyretin tetramerization. J. Am. Chem. Soc. 125:13404, 2003.

Huff, M.E., Balch, W.E., Kelly, J.W. Pathological and functional amyloid formation orchestrated by the secretory pathway. Curr. Opin. Struct. Biol. 13:674, 2003.

Huff, M.E., Page, L.J., Balch, W.E., Kelly, J.W. Gelsolin domain 2 Ca2+ affinity determines susceptibility to furin proteolysis and familial amyloidosis of Finnish type. J. Mol. Biol. 334:119, 2003.

Hurshman, A.R., White, J.T., Powers, E.T., Kelly, J.W. Transthyretin aggregation under partially denaturing conditions is a downhill polymerization. Biochemistry 43:7365, 2004.

Kustedjo, K., Deechongkit, S., Kelly, J.W., Cravatt, B.F. Recombinant expression, purification, and comparative characterization of torsinA and its torsion dystonia-associated variant ΔE-torsinA. Biochemistry 42:15333, 2003.

Miller, S.R., Sekijima, Y., Kelly, J.W. Native state stabilization by NSAIDs inhibits transthyretin amyloidogenesis from the most common familial disease variants. Lab. Invest. 84:545, 2004.

Powers, E.T., Powers, D.L. A perspective on mechanisms of protein tetramer formation. Biophys. J. 85:3587, 2003.

Reixach, N., Deechongkit, S., Jiang, X., Kelly, J.W., Buxbaum, J.N. Tissue damage in the amyloidoses: transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture. Proc. Natl. Acad. Sci. U. S. A. 101:2817, 2004.

You, S.L., Kelly, J.W. Highly efficient biomimetic total synthesis and structural verification of bistratamides E and J from Lissoclinum bistratum. Chemistry 10:71, 2004.

You, S.L., Kelly, J.W. Highly efficient enantiospecific synthesis of imidazoline-containing amino acids using bis(triphenyl)oxodiphosphonium trifluoromethanesulfonate. Org. Lett. 6:1681, 2004.

You, S.L., Kelly, J.W. Total synthesis of dendroamide A: oxazole and thiazole construction using an oxodiphosphonium salt. J. Org. Chem. 68:9506, 2003.

Zhang, Q., Kelly, J.W. Cys10 mixed disulfides make transthyretin more amyloidogenic under mildly acidic conditions. Biochemistry 42:8756, 2003.

Zhang, Q., Powers, E.T., Nieva, J., Huff, M.E., Dendle, M.A., Bieschke, J., Glabe, C. G., Eschenmoser, A., Wentworth, P., Jr., Lerner, R.A., Kelly, J.W. Metabolite-initiated protein misfolding may trigger Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 101:4752, 2004.


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

Kelly Web Site