News and Publications

Bioorganic Chemistry and Neurochemistry

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 in cells.

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.

Publications

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. 62:2259, 1997.

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, 1997.

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 press.