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The Skaggs Institute
for Chemical Biology


Scientific Report 2007




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.

 

Jeffery W. Kelly, Ph.D.
Lita Annenberg Hazen Professor of Chemistry

Dean, Graduate and Postgraduate Studies

Kelly Web Site