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. DHaeze, 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
goal is to understand molecular mechanisms of protein folding and of protein misfolding, which
leads to neurodegenerative diseases, including Alzheimers and Parkinsons 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.
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
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 reticulumassisted folding determines protein secretion
in a tissue-specific manner, and we propose that its competition with endoplasmic reticulumassociated
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
accelerating the amyloidogenesis of these peptides, which are associated with Alzheimers
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.
including Parkinsons disease and dementia with Lewy bodies, are characterized by cytoplasmic
aggregates within degenerating dopaminergic neurons in the substantia nigra. Clinical observations
suggest a correlation between oxidative stress or inflammation and Parkinsons 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.
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 23231 forms a mixture of monomers, dimers, and trimers,
whereas PrP 90231, 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 23231 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 90231 and PrP 23231; however, monomer depletion occurs much more slowly for
PrP 90231. We also observed that if a second stage of aggregation occurs, it must occur in
a very inefficient way. The aggregates formed by PrP 90231 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 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
Alzheimers 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,
Deechongkit, S., Powers, E.T.,
You, S.-L., Kelly, J.W. Controlling the morphology of cross
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,
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.
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.
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.