The Skaggs Institute
for Chemical Biology
Understanding the Mechanisms of Protein Folding and Misfolding/Misassembly
J.W. Kelly, J. Bieschke, D.A. Bosco,
E. Culyba, M.T.A. Dendle, W. DHaeze, 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
on protein folding and misfolding will enable us to design new therapeutic strategies
to ameliorate protein misfolding/misassembly diseases, such as Alzheimers,
Parkinsons, 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
Aggregation of Amyloid β-Peptide Associated with AlzheimerS 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 Alzheimers 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 Alzheimers disease in which aging was delayed by diminished insulin/insulin
growth factor-1like 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
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 reticulumassociated
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 domains 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.
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 Aβ140
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-olefinAβanalog 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 Alzheimers disease produce positive results, our findings are important
analogs can be used as an antigen to elicit an immune response against a specific
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.
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 Alzheimers 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., DHaeze, 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., DHaeze, 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