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The Skaggs Institute
for Chemical Biology

Scientific Report 2006

Understanding the Mechanisms of Protein Folding and Misfolding/Misassembly

J.W. Kelly, J. Bieschke, D.A. Bosco, E. Culyba, M.T.A. Dendle, W. D’Haeze, T.R. Foss, D.M. Fowler, Y. Fu, A. Fuller, J. Gao, M.Y. Gao, S.M. Johnson, T. Mu, A. Murray, E.T. Powers,P. Rao, A.R. Sawkar, L. Segatori, S. Siegel, J.Y. Suk, K. Usui, R.L. Wiseman, I. Yonemoto, Z. Yu

Mechanistic studies on protein folding and misfolding will enable us to design new therapeutic strategies to ameliorate protein misfolding/misassembly diseases, such as Alzheimer’s, Parkinson’s, and Gaucher diseases. This goal will be accomplished by using organismal and cell biological disease models as well as spectroscopic and biophysical approaches in combination with chemical synthesis and molecular biology. Maintaining critical collaborations with W.E. Balch and J. Buxbaum, Scripps Research, and A. Dillin, the Salk Institute for Biological Studies, La Jolla, California, is pivotal to the success of the projects described here.

Functional Amyloid in Mammalian Tissue

Melanocytes, highly specialized mammalian cells present in the skin and eye, contain melanosomes, membrane-delimited organelles characterized by the presence of relatively high amounts of melanin. Melanin functions as a protectant against pathogens, oxidative damage, and, especially, ultraviolet radiation. It has been reported that maturation of the melanosome involves the production of Pmel17 fibers. We hypothesized that these fibers had an amyloid structure.

Amyloid formation is generally associated with the onset of neurodegenerative diseases; however, we showed for the first time that functional amyloid exists in mammalian cells. Formation of Pmel17 amyloid is mediated by the secretory pathway and is required for and accelerates the polymerization of reactive small molecules into melanin. The presence of Pmel17 amyloid also decreases the diffusion of toxic melanin precursor molecules out of the melanosome. In vitro fibril formation with recombinant Pmel17 occurs with an unprecedented rapidity, suggesting that this process has been optimized throughout evolution to avoid toxic amyloidogenic intermediates. Efficient formation of functional Pmel17 amyloid protects cells against the toxic intermediates formed during the synthesis of melanin.

The discovery of functional Pmel17 amyloid in mammalian cells strongly suggests the presence of other amyloids with distinct and well-defined functions that still need to be identified. Comparing pathologic amyloid with functional amyloid should help us appreciate why the former type leads to neurodegeneration.

Aggregation of Amyloid β-Peptide Associated with Alzheimer’S Disease and Disaggregation

In collaboration with P. Wentworth and R.A. Lerner, Scripps Research, we showed that oxidative cholesterol metabolites can covalently modify amyloid β-peptides (Aβ), dramatically accelerating the amyloidogenicity of these peptides, which are associated with Alzheimer’s disease. Metabolite-initiated Aβ amyloidogenesis occurs via a 2-step mechanism that involves the energetically downhill assembly of spherical aggregates by Aβ-metabolite adducts before the generation of fibrillar aggregates.

In collaboration with Dr. Dillin, we suggested that a mechanistic link exists between aging and aggregation-mediated proteotoxic effects. The toxic effects related to Aβ aggregation were substantially reduced in a Caenorhabditis elegans model of Alzheimer’s disease in which aging was delayed by diminished insulin/insulin growth factor-1–like signaling, a pathway essential to the longevity and youthfulness of worms and other eukaryotic organisms. Two downstream transcription factors regulate opposing disaggregation and aggregation activities to ensure the protection of cells against the toxic effects of Aβ aggregation. In an ongoing project, we are screening for small molecules that slow aging in the C elegans model system to decrease the toxic effects of Aβ and other types of aggregates and enhance the activity of these protective pathways.

Chemical Chaperones to Ameliorate Gaucher Disease

Mutations in the gene that encodes glucocerebrosidase, a lysosomal hydrolase, lead to an accumulation of the glucocerebrosidase substrate, glucosylceramide, in the lysosome, causing Gaucher disease, one of the most common lysosomal storage disorders. Previously, we showed that small molecules such as N-(n-nonyl)-
deoxynojirimycin increase the activity of the glucocerebrosidase variant N370S in a cell line derived from tissue from a patient with Gaucher disease. This “chemical chaperoning” effect most likely is due to the binding of the small molecule to the native state of N370S, which stabilizes the glucocerebrosidase and allows it to traffic from the endoplasmic reticulum to the lysosomes. Interestingly, the activity of G202R glucocerebrosidase, a variant retained in the endoplasmic reticulum, is also increased in the presence of chemical chaperones.

In situ localization studies illustrated that the cellular localization pattern of the N370S, L444P, and G202R glucocerebrosidase variants is distinct and can be manipulated in the presence of chemical chaperones. N370S, L444P, and G202R are destabilized in the neutral pH environment of the endoplasmic reticulum, making them susceptible to endoplasmic reticulum–associated degradation or retention. The instability of N370S and G202R in the endoplasmic reticulum is corrected by the presence of chemical chaperones.

Our results show that chemical chaperones enhance the activity of distinct glucocerebrosidase variants to an extent that may be sufficient to ameliorate Gaucher disease. Preliminary data suggest that most likely certain glucocerebrosidase mutants (e.g., L444P) will require the design of specific chemical chaperones that target the compromised domain in order to facilitate proper trafficking and partial restoration of the domain’s function.

We are using Gaucher disease as a model system to screen for small-molecule modulators of protein folding that would influence the biological machinery that affects cellular protein folding and export from the endoplasmic reticulum. Ideally, such molecules would enhance the ability of the cellular protein folding machinery to fold and secrete destabilized proteins, including glucocerebrosidase variants, and would therefore be useful to ameliorate a variety of loss-of-function protein misfolding diseases.
β-Sheet Folding

Recently, we reengineered the loop 1 substructure of the PIN WW domain, a 34-residue protein composed of 3 β-strands and 2 intervening loops. The folding of loop 1 is the rate-limiting step for folding of the PIN WW domain. Replacement of the wild-type loop 1 structure with engineered shorter sequences hastened WW domain folding. However, the accelerated folding was accompanied by an elimination of WW domain function. This finding strongly suggests that the loop 1 sequence has been optimized throughout evolution for WW domain function but not for WW domain folding.

We also developed an approach to synthesize significant amounts of dipeptide isosteres required to introduce an E-olefin perturbation in a given polypeptide backbone. Unlike amide-to-ester alterations, E-olefin perturbations do not introduce unfavorable electrostatic interactions when perturbing hydrogen bonding. This approach was first applied to the Aβ1–40 peptide in which a phenylalanine-phenylalanine E-olefin dipeptide isostere was incorporated to replace the 2 phenylalanine residues at positions 19 and 20, perturbing hydrogen bonds in Aβ fibrils. In contrast to wild-type Aβ1-40, the resulting E-olefin–Aβanalog was unable to form fibrils and exclusively formed spherical aggregates that could assemble into larger amorphous aggregates. These observations indicate that the removal of 2 hydrogen bonds prevents the formation of Aβ fibrils but does not affect the formation of spherical aggregates. If immunization trials for Alzheimer’s disease produce positive results, our findings are important because E-olefin–Aβ analogs can be used as an antigen to elicit an immune response against a specific toxic aggregate.

Besides understanding the role of particular hydrogen bonds for proper protein folding, the ultimate aim of this research is to design and synthesize proteomimetics that fold and function despite having significantly fewer amide bonds than their parental counterparts and that have an improved membrane permeability that could enable oral bioavailability.


Bieschke, J., Zhang, Q., Bosco, D.A., Lerner, R.A., Powers, E.T., Wentworth, P., Jr., Kelly, J.W. Small molecule oxidation products trigger disease-associated protein misfolding. Acc. Chem. Res. 39:611, 2006.

Bosco, D.A., Fowler, D.M., Zhang, Q., Nieva, J., Powers, E.T., Wentworth, P., Jr., Lerner, R.A., Kelly, J.W. Elevated levels of oxidized cholesterol metabolites in Lewy body disease brains accelerate α-synuclein fibrilization [published correction appears in Nat. Chem. Biol. 2:346, 2006]. Nat. Chem. Biol. 2:249, 2006.

Cohen, E., Bieschke, J., Perciavalle, R.M., Kelly, J.W., Dillin, A. Opposing activities protect against age-onset proteotoxicity. Science 313:1604, 2006.

Cordeiro, Y., Kraineva, J., Suarez, M.C., Tempesta, A.G., Kelly, J.W., Silva, J.L., Winter, R., Foguel, D. Fourier transform infrared spectroscopy provides a fingerprint for the tetramer and for the aggregates of transthyretin. Biophys. J. 91:957, 2006.

Deechongkit, S., Nguyen, H., Jäger, M., Powers, E.T., Gruebele, M., Kelly, J.W. β-Sheet folding mechanisms from perturbation energetics. Curr. Opin. Struct. Biol. 16:94, 2006.

Foss, T.R., Wiseman, R.L., Kelly, J.W. The pathway by which the tetrameric protein transthyretin dissociates. Biochemistry 44:15525, 2005.

Fowler, D.M., Koulov, A.V., Alory-Jost, C., Marks, M.S., Balch, W.E., Kelly, J.W. Functional amyloid formation within mammalian tissue. PLoS Biol. 4:e6, 2006.

Fu, Y., Bieschke, J., Kelly, J.W. E-Olefin dipeptide isostere incorporation into a polypeptide backbone enables hydrogen bond perturbation: probing the requirements for Alzheimer’s amyloidogenesis. J. Am. Chem. Soc. 127:15366, 2005.

Green, N.S., Foss, T.R., Kelly, J.W. Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis. Proc. Natl. Acad. Sci. U. S. A. 102:14545, 2005.

Jäger, M., Zhang, Y., Bieschke, J., Nguyen, H., Dendle, M., Bowman, M.E., Noel, J.P., Gruebele, M., Kelly, J.W. Structure-function-folding relationship in a WW domain. Proc. Natl. Acad. Sci. U. S. A. 103:10648, 2006.

Johnson, S.M., Wiseman, R.L., Sekijima, Y., Green, N.S., Adamski-Werner, S.L., Kelly, J.W. Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a focus on the transthyretin amyloidoses. Acc. Chem. Res. 38:911, 2005.

Kelly, J.W., Balch, W.E. The integration of cell and chemical biology in protein folding. Nat. Chem. Biol. 2:224, 2006.

Page, L.J., Suk, J.Y., Huff, M.E., Lim, H.-J., Venable, J., Yates, J. III, Kelly, J.W., Balch, W.E. Metalloendoprotease cleavage triggers gelsolin amyloidogenesis. EMBO J. 24:4124, 2005.

Powers, E.T., Deechongkit, S., Kelly, J.W. Backbone-backbone H-bonds make context-dependent contributions to protein folding kinetics and thermodynamics: lessons from amide-to-ester mutations. Adv. Protein Chem. 72:39, 2005.

Sawkar, A.R., Adamski-Werner, S.L., Cheng, W.-C., Wong, C.-H., Beutler, E., Zimmer, K.-P., Kelly, J.W. Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles. Chem. Biol. 12:1235, 2005.

Sawkar, A.R., D’Haeze, W., Kelly, J.W. Therapeutic strategies to ameliorate lysosomal storage disorders: a focus on Gaucher disease. Cell. Mol. Life Sci. 63:1179, 2006.

Sawkar, A.R., Schmitz, M., Zimmer, K.-P., Reczek, D., Edmunds, T., Balch, W.E., Kelly, J.W. Chemical chaperones and permissive temperatures alter localization of Gaucher disease associated glucocerebrosidase variants. ACS Chem. Biol. 1:235, 2006.

Sekijima, Y., Dendle, M.T., Kelly, J.W. Orally administered diflunisal stabilizes transthyretin against dissociation required for amyloidogenesis. Amyloid 13:236, 2006.

Sekijima, Y., Dendle, M.T., Wiseman, R.L., White, J.T., D’Haeze, W., Kelly, J.W. R104H may suppress transthyretin amyloidogenesis by thermodynamic stabilization, but not by the kinetic mechanism characterizing T119 interallelic trans-suppression. Amyloid 13:57, 2006.

Sörgjerd, K., Ghafouri, B., Jonsson, B.-H., Kelly, J.W., Blond, S.Y., Hammarström, P. Retention of misfolded mutant transthyretin by the chaperone BiP/GRP78 mitigates amyloidogensis. J. Mol. Biol. 356:469, 2006.

Suk, J.Y., Zhang, F., Balch, W.E., Linhardt, R.J., Kelly, J.W. Heparin accelerates gelsolin amyloidogenesis. Biochemistry 45:2234, 2006.

Wiseman, R.L., Powers, E.T., Kelly, J.W. Partitioning conformational intermediates between competing refolding and aggregation pathways: insights into transthyretin amyloid disease. Biochemistry 44:16612, 2005


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

Dean, Graduate and Postgraduate Studies

Kelly Web Site