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
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-sheetrich,
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
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
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
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.
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 Alzheimers disease. Proc. Natl. Acad. Sci. U. S. A. 101:4752, 2004.