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The Skaggs Institute
for Chemical Biology
Understanding the Mechanisms of Protein Folding and Misfolding/Misassembly
J.W. Kelly, S. Choi, E. Culyba, D. Du, M.T.A. Dendle, W. D'Haeze, D.M. Fowler,
A. Fuller, J. Gao, M.Y. Gao, T. Mu, A. Murray, E.T. Powers, P. Rao, M. Saure, L. Segatori, S. Siegel, J.Y. Suk, K. Usui, M. Wang, I. Yonemoto, Z. Yu, J. Zhu
We
study the mechanisms of protein folding and misfolding to gain insight that can
be used to develop new therapeutic strategies for diseases due to protein misfolding/misassembly,
such as Alzheimer's, Parkinson's, and Gaucher diseases. We use biophysical
approaches in combination with organismal and cell biological disease models to
accomplish this goal. Our work benefits greatly from our collaborations with J.
Buxbaum and W.E. Balch, Scripps Research, and A. Dillin, Salk Institute for Biological
Sciences, La Jolla, California.
Effect of Oxidative Metabolites on Aggregation of β-Amyloid Peptide Associated with Alzheimer's Disease
In collaboration
with P. Wentworth and R.A. Lerner, Skaggs Institute, we showed that aldehyde-containing
oxidative metabolites of cholesterol covalently modify β-amyloid
peptides (Aβ),
thereby dramatically accelerating the aggregation of Aβ,
a process central to the pathogenesis of Alzheimer's disease. We have now shown
that another common oxidative metabolite of lipids, 4-hydroxynonenal, also accelerates
Aβ
aggregation through covalent modification. This metabolite contains an α,β
unsaturated aldehyde that can react with proteins by 1,4 conjugate addition, Schiff
base formation, or both. Patients with Alzheimer's disease have elevated levels
of 4-hydroxynonenal, and it has been detected in deposits of Aβ
aggregates found in post mortem samples of the brains of patients with the disease.
We have shown that 4-hydroxynonenal covalently modifies Aβ
at lysine and histidine residues and that because it is bifunctional, it can also
cross-link Aβ
molecules. The consequence of these reactions is that 4-hydroxynonenal accelerates
the formation of small, protofibrillar Aβ
aggregates while inhibiting the formation of longer, mature fibrils. Data accumulated
by many groups during the past several years suggest that small aggregates of the
type that Aβ
forms under the influence of 4-hydroxynonenal probably are important in the pathogenesis
of Alzheimer's disease.
Chemical Chaperones to Ameliorate Gaucher Disease
Mutations in
the gene for glucocerebrosidase, a lysosomal hydrolase, result in lysosomal accumulation
of its substrate, glucosylceramide, causing Gaucher disease, one of the most common
lysosomal storage disorders. Previously, we showed that small molecules that bind
to the active site of glucocerebrosidase increase the activity of N370S glucocerebrosidase
in a cell line derived from tissue from a patient with Gaucher disease, even though
the molecules inhibit the enzyme in vitro. This chemical chaperoning
effect is caused by binding of the small molecule to the native state of N370S variant
glucocerebrosidase; this binding stabilizes the enzyme, allowing it to traffic from
the endoplasmic reticulum to its target destination, the lysosome, instead of being
degraded.
The results
of our studies on structure-activity relationships during the past several years
have enabled us to identify substructures that enhance binding to glucocerebrosidase.
In the past year, we combined the structure-activity data from different chemical
series to produce potent novel chaperones composed of a carbohydrate-like substructure
that binds in the active site and a hydrophobic substructure that binds in a nearby
pocket. Specifically, we joined isofagomine and a glucitol derivative with adamantyl
amides through linkers of various lengths. The most potent compounds increased the
activity of N370S and G202R glucocerebrosidases by 2.5- and 7.2-fold, respectively,
in cell lines derived from the tissues of patients with Gaucher disease. These increases
are the best we have observed to date, making the compounds discovered in this study
excellent leads for the development of Gaucher disease therapeutics.
Amide-to-E-Olefin vs Amide-to-Ester Backbone Perturbations
Deciphering
the contributions of backbone-backbone hydrogen bonding to the energetics of protein
folding is an area of great interest. Because backbone hydrogen bonds occur between
main-chain amides, the bonds can be perturbed by replacing the amide bond of interest
in a protein with an isostructural moiety with reduced or nonexistent hydrogen-bonding
capacity. Currently, the most convenient way to perturb backbone hydrogen bonding
is to replace amides with esters. Esters lack a hydrogen-bond donor and are weaker
hydrogen-bond acceptors than are amides but are otherwise similar. Esters are therefore
excellent hydrogen bond–perturbing replacements for amides.
In one approach,
termed amide-to-ester (A-to-E) mutation, an electrostatic repulsion is introduced
between the ester O replacing the NH and the carbonyl oxygen of the acceptor amide.
The magnitude of this O-O repulsion is unknown, complicating the extraction of hydrogen-bond
energies from A-to-E perturbations.
It has been
proposed that an amide-to-E-olefin (A-to-O) mutation may be an even better
hydrogen bond–perturbing mutation than A-to-E mutation. A-to-O mutations eradicate
both the hydrogen-bond donating and accepting capacity of the protein backbone without
introducing extraneous electrostatic repulsions. However, introducing A-to-O mutations
into proteins is challenging because it is difficult to stereospecifically synthesize
the necessary alkene-containing dipeptide isosteres for incorporation into the peptide/protein
sequence.
We previously
developed methods to synthesize the phenylalanine-phenylalanine E-olefin
dipeptide isostere and incorporate it into peptides. We used this technology in
this past year to introduce an A-to-O mutation between residues 22 and 23 of the
Pin WW domain, a well-studied 3-stranded β-sheet
protein domain. Comparing the effect of this mutation on the folding thermodynamics
of the Pin WW domain with the effects of the analogous A-to-E mutation enabled us
to quantify the repulsive O-O interaction introduced by A-to-E mutations and to
establish the energy of the hydrogen bond formed by the backbone amide NH of residue
23.
Both A-to-E
and A-to-O mutations delete the same hydrogen bond in the hydrophobic core and lead
to a pronounced decrease in protein stability. The folding free energies of the
ester and olefin mutants together with the transfer free energies measured on relevant
model compounds provide an estimate of 0.3 kcal/mol for the O-O electrostatic repulsion
term in the context of a β-sheet
hydrogen-bond network. Knowing this value should enable more accurate measurements
of the strength of hydrogen bonds by using A-to-E mutations. For example, from these
data, the hydrogen bond between phenylalanine at position 23 and arginine at position
14 in the Pin WW domain stabilizes the folded state by 1.3 kcal/mol.
β-Sheet Folding
We have for
many years used an N-terminally truncated yet cooperatively folded version of the
human Pin1 WW domain (residues 6–39) as a model system for understanding β-sheet
folding energetics. During the past year, we found that the negatively charged N-terminal
sequence (Met-Ala-Asp-Glu-Glu), which we had previously deleted and which is not
conserved in other WW domain family members, stabilizes the human Pin1 WW domain
by approximately 4 kJ mol-1 at 65°C. N-terminal truncations and
mutations in conjunction with a double-mutant cycle analysis and a recently published
high-resolution x-ray structure of full human Pin1 protein suggest that an ionic
interaction between the negatively charged residues in the N-terminal tail and the
side chain of lysine 13 in the first β-strand
causes the observed increase in stability.
Ligand-binding
studies indicate that the ionic interaction between lysine 13 and the charged N
terminus is the best solution for enhancing WW domain stability without compromising
function. However, kinetic laser temperature-jump relaxation studies revealed that
this stabilizing interaction does not exist in the folding transition state near
physiologic temperature. This observation indicates that the negatively charged
N-terminal sequence contributes differently to protein stability and folding rate.
Our data further suggest that caution must be exercised when choosing domain boundaries
for WW domains (and indeed all other proteins) for structural, functional, or thermodynamic
studies.
Publications
Dillin, A., Kelly, J.W. The yin-yang of sirtuins. Science 317:461, 2007.
Fowler, D.M., Koulov, A.V., Balch, W.E., Kelly, J.W. Functional amyloid: from bacteria to humans. Trends Biochem. Sci. 32:217, 2007.
Fu, Y., Gao, J., Bieschke, J., Dendle, M.A., Kelly, J.W. Amide-to-E-olefin versus amide-to-ester backbone H-bond perturbations: evaluating the O-O repulsion
for extracting H-bond energies. J. Am. Chem. Soc. 128:15948, 2006.
Jäger, M., Dendle, M., Kelly, J.W. A cross-strand Trp-Trp pair stabilizes the hPin1 WW domain at the expense of function.
Protein Sci. 16:2306, 2007.
Jäger, M., Nguyen, H., Dendle, M., Gruebele, M., Kelly, J.W. Influence of hPin1 WW N-terminal domain boundaries on function, protein stability,
and folding. Protein Sci. 16:1495, 2007.
Kelly, J.W. A biochemist considers whether protein misfolding plays a part in type II diabetes [Research Highlights/Journal Club]. Nature 446:113, 2007.
Münch, J., Rücker, E., Ständker, L., Adermann, K., Goffinet, C., Schindler, M., Wildum, S., Chinnadurai, R., Rajan, D., Specht, A., Giménez-Gallego, G., Sánchez,
P.C., Fowler, D.M., Koulov, A., Kelly, J.W., Mothes, W., Grivel, J.C., Margolis, L., Keppler, O.T., Forssmann, W.G., Kirchhoff, F. Semen-derived
amyloid fibrils dramatically enhance HIV infection. Cell 131:1059, 2007.
Reixach, N., Adamanski-Werner, S.L., Kelly, J.W., Koziol, J., Buxbaum, J.N. Cell-based screening of inhibitors of transthyretin aggregation. Biochem. Biophys.
Res. Commun. 348:889, 2006.
Siegel, S.J., Bieschke, J., Powers, E.T., Kelly, J.W. The oxidative stress metabolite 4-hydroxynonenal promotes Alzheimer protofibril formation.
Biochemistry 46:1503, 2007.
Stewart, C.R., Wilson, L.M., Zhang, Q., Pham, C.L.L., Waddington, L.J., Staples, M.K., Stapleton, D. Kelly, J.W., Howlett, G.J. Oxidized
cholesterol metabolites found in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibril formation. Biochemistry 46:5552, 2007.
Tojo, K., Sekijima, Y., Kelly, J.W., Ikeda, S.-I. Diflunisal stabilizes familial amyloid polyneuropathy-associated transthyretin variant
tetramers in serum against dissociation required for amyloidogenesis. Neurosci. Res. 56:441, 2006.
Wang, X., Venable, J., LaPointe, P., Hutt, D.M., Koulov, A.V., Coppinger, J., Gurkan, C., Kellner, W., Matteson, J., Plutner, H., Riordan, J.R., Kelly, J.W., Yates, J.R.
III, Balch, W.E. Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 127:803, 2006.
Wiseman, .L., Koulov, A., Powers, E.T., Kelly, J.W., Balch, W.E. Protein energetics in maturation of the early secretory pathway. Curr. Opin. Cell
Biol. 19:359, 2007.
Yu, Z, Sawkar, A.R., Kelly, J.W. Pharmacologic chaperoning as a strategy to treat Gaucher disease. FEBS J. 274:4944, 2007.
Yu, Z., Sawkar, A.R., Whalen, L.J., Wong, C.-H., Kelly, J.W. Isofagomine and 2,5-anhydro-2,5-imino-D-glucitol-based glucocerebrosidase pharmacological
chaperones for Gaucher disease intervention. J. Med. Chem. 50:94, 2007.
Zhang, Y., Kim, Y., Genoud, N., Gao, G., Kelly, J.W., Platt, S.N., Gill, G., Dixon, J.E., Noel, J.P. Determinants for dephosphorylation of the RNA polymerase II C-terminal domain by Scp-1. Mol.
Cell 24:759, 2006.
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