News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998
Bioorganic Chemistry and Neurochemistry
J.W. Kelly, H. Bekele, E. Koepf, H. Lashuel, A. Lentz, V. Oza, M. Petrassi,
E. Powers, H. Purkey, P. Raman, G. Ratnaswamy, T. Walkup, L. Woo, Y. Xie
The central theme of our research is to understand the chemistry and biology
of peptides and proteins and to develop new approaches for manipulating these
properties with purposefully designed small molecules. Under the auspices of
The Skaggs Institute of Chemical Biology, we are focusing our research on neurodegenerative
diseases. Our goal is to learn enough about the mechanism of these diseases to
develop novel therapeutic strategies.
Mechanism of Huntington's Disease
Huntington's disease is referred to as a triplet-repeat disease because the
number of consecutive CAG codons in the gene coding for the 3100 amino acid protein
huntingtin increases from 21 to more than 39, and that increase alone brings
on the disease. In fact, expression of just the CAG-encoded part of the protein
with the more than 39 glutamine residues (CAG codes for glutamine) in mice reproduces
nearly all aspects of the human disease. Both mice and humans have amyloid-like
polyglutamine fibrils in the nucleus of neurons that appear to cause the disease.
This finding is interesting, because huntingtin is a cytoplasmic protein, not
a nuclear protein.
We are addressing the following issues: (1) What is the structure of the
glutamine polypeptides in water, and how can this structure help explain the
observed pathologic changes? (2) Does the expanded glutamine sequence act as
a nuclear localization sequence, a situation that would explain how this protein
enters the nucleus? (3) Because the glutamine polypeptides make some kind of
aggregated ß-sheet structure in water, we are developing a genetic selection
system to detect molecules that can prevent assembly of the sheets, the putative
cause of the disease.
Conversion of a Normally Soluble and Functional Human Protein Into Amyloid Fibrils
An invariant feature of the 16 human amyloid diseases (e.g., Alzheimer's
disease) is the presence of amyloid fibrils near dead or dying neurons. We are
studying 3 amyloidogenic proteins: transthyretin, gelsolin, and amyloid-ß.
Conversion of these proteins from a well-defined tertiary structure into a quaternary
structure rich in ß-sheets appears to be the causative event in the diseases
associated with the proteins (Fig. 1).
We use analytical ultracentrifugation and mass spectrometry to study the
changes in quaternary structure and circular dichroism, fluorescence, and nuclear
magnetic resonance spectroscopy to investigate the changes in tertiary structure
required for formation of amyloid fibrils. We use electron and atomic force microscopy
to examine the mechanism of assembly of the amyloidogenic intermediate and hydrogen-deuterium
exchange with electrospray and matrix-assisted laser desorption/ionization mass
spectrometry to determine dynamic characteristics of the intermediate.
We now know that significant changes in tertiary structure are required for
the conversion of these proteins into amyloid. The challenge remaining is to
understand the details of these changes, the dynamic properties of the intermediates,
and the self-assembly mechanism that yields amyloid.
Neurotoxicity of Amyloid and the Soluble Amyloid Precursors
Neurotoxic effects, which are a common feature of all amyloid diseases and
of Huntington's disease, are poorly understood. Most likely amyloid causes toxic
effects through multiple mechanisms. We have embarked on a project to understand
why amyloid fibrils kill nerve cells. Specifically, we have prepared fluorescently
labeled amyloid and glutamine polypeptides to understand the fate of these molecules
Prevention of Conformational Changes Associated With Human Amyloid Disease
We introduced a new therapeutic strategy in which a high-affinity ligand
is used to prevent the quaternary and/or tertiary structural changes required
for the formation of amyloid fibrils. In this project, we use x-ray crystallography
or nuclear magnetic resonance spectroscopy in combination with synthetic chemistry
to discover high-affinity inhibitors. Recent cocrystal structures of the normally
folded form of transthyretin with second-generation inhibitors suggest new molecules
to synthesize and test.
Our philosophy is to develop simple parallel syntheses that can be used to
prepare libraries to quickly sort out structure-activity relationships. The genetic
selection system under development for the detection of inhibitors of polyglutamine
aggregation and an increasing synthetic effort should provide therapeutic solutions
to Huntington's disease, which may also be an amyloid-like disease.
Bekele, H., Nesloney, C.L., McWilliams, K.W., Zacharius, N.M., Chitnumsub,
P., Kelly, J.W. Improved synthesis of the Boc and Fmoc derivatives of 4-(2´-aminoethyl)-6-dibenzofuranpropionic
acid: An unnatural amino acid that nucleates beta-sheet folding. J. Org. Chem.
Chen, H.I., Einbond, A., Kwak, S.-J., Linn, H., Koepf, E., Peterson, S.,
Kelly, J.W., Sudol, M. Characterization of the WW domain of the human Yes-associated
protein and its polyproline-containing ligands. J. Biol. Chem. 272:17070, 1997.
Colon, W., Lai, Z., Lashuel, H.A., McCulloch, J., McCutchen, S.L., Miroy,
G.J., Peterson, S.A., Kelly, J.W. Transthyretin quaternary and tertiary structural
changes facilitate misassembly into amyloid: A new therapeutic strategy based
on preventing the amyloidogenic conformational changes. Adv. Protein Chem. 50:161,
Dikler, S., Kelly, J.W., Russell, D.H. Improving mass spectrometric
sequencing of arginine-containing peptides by derivatization with acetylacetone.
J. Mass. Spectrom. 32:1337, 1997.
Kelly, J.W. The alternative conformations of amyloidogenic proteins
and their multi-step assembly pathways. Curr. Opin. Struct. Biol. 8:101, 1998.
Kelly, J.W. Amyloid fibril formation and protein misassembly: A structural
quest for insights into amyloid and prion diseases. Structure 5:595, 1997.
Kelly, J.W. The environmental dependency of protein folding best explains
prion and amyloid diseases. Proc. Natl. Acad. Sci. U.S.A. 95:930, 1997.
Lai, Z., McCulloch, J., Kelly, J.W. GdnHCl-Induced denaturation and
refolding of transthyretin exhibits a marked hysteresis: Equilibria with high
kinetic barriers. Biochemistry 36:10230, 1997.
Nettleton, E.J., Sunde, M., Lai, Z., Kelly, J.W., Dobson, C.M., Robinson,
C.V. Protein subunit interactions and structural integrity of amyloidogenic
transthyretins: Evidence from electrospray mass spectrometry. J. Mol. Biol., in