Scientific Report 2007

Chemistry

Chemistry, Biology, and Inflammatory Disease

P. Wentworth, Jr., J.Y. Chang, Y.P. Chen, J. Dambacher, R.K. Grover, J. Nieva, M. Puga, B.D. Song, M.M.R. Peram, J. Rogel, S.R. Troseth, H. Wang, A.D. Wentworth

Our research is interdisciplinary and involves aspects of bioorganic, biophysical, physical organic, synthetic, and analytical chemistry coupled with biochemical techniques, cell-based assays, and animal models. Ongoing projects include studies on atherosclerosis, neurodegenerative diseases, ischemia-reperfusion injury, macular degeneration, cancer, inflammation, and infectious diseases.

The Antibody-Catalyzed Water Oxidation Pathway

Our discovery that all antibody molecules, regardless of source or antigenic specificity, can catalyze the reaction between singlet oxygen and water to give hydrogen peroxide is causing a revision of the axiom that antibodies are the classical adapter molecule of the immune system, linking recognition and killing of foreign pathogens. Both the chemical and the biological aspects of this pathway are being explored extensively, and intriguing new insights into how this pathway may play a role in immune defense and inflammatory damage are emerging.

We are searching for the active site for the antibody-catalyzed water oxidation pathway within the antibody structure. We have cloned and expressed soluble individual domains (V_HV_L, C_H1C_L, V_H, V_L, CH₁, C_L) of the mouse Fab 4C6. All the domains have been successfully purified to homogeneity from the periplasm of Escherichia coli transformed with plasmids encoding individual domains. All the domains are folded, as indicated by ultraviolet circular dichroism. These findings indicate that all these immunoglobulin domains can participate in the antibody-catalyzed water oxidation pathway when activated with singlet dioxygen.

Inflammatory Aldehydes and Protein Misfolding

We have shown that the inflammation-derived cholesterol seco-sterols atheronal-A and atheronal-B (Fig. 1) trigger a deformation in the secondary structure of the normally folded low-density lipoprotein apoB-100 into a proamyloidogenic form. In collaboration with J.W. Kelly and colleagues, Department of Chemistry, we extended this model and showed that these cholesterol seco-sterols also trigger the misfolding of amyloid β-peptide_1–40, leading to formation of fibrils similar to those observed in patients with Alzheimer's disease. More recently, using mutated synthetic sequences of amyloid β-peptide_1–40, we found that the accelerated aggregation of this protein only occurs when a particular lysine of the sequence is modified. We have also shown that atheronals and other lipid aldehydes accelerate the aggregation of several wild-type amyloidogenic proteins, including immunoglobulin light chains, mouse prions, and the tumor suppressor protein p53. The generality and specificity of this process suggest that inflammatory aldehydes and their posttranslational modification of amyloidogenic peptides may be the chemical link between the known associations of inflammation, oxidative damage, and various protein misfolding diseases.

Fig. 1. The cholesterol seco-sterols atheronal-A (top) and atheronal-B (bottom).

Interaction Between Protozoan J-binding Protein 1 and Glycosylated DNA

Current treatments of parasitic infections such as leishmaniasis (cutaneous or visceral, Leishmania species), African trypanosomiasis (sleeping sickness, Trypanosoma brucei), and American trypanosomiasis (Chagas disease, Trypanosoma cruzi) have limited effectiveness, increasing drug resistance and the inherent toxic effects of the drugs. Thus, an elucidation of new parasite-specific biological targets for therapeutic agents is needed. In this regard, the discovery that DNA from members of the order Kinetoplastida, but not from other eukaryotes, contains an unusual modified base, β-D-glucosyl(hydroxymethyl)uracil, called base J was a breakthrough. Extracts of several kinetoplastids contain a J-binding protein (JBP) that specifically binds to J-containing duplex DNA. JBP-1 is essential in Leishmania.

As a drug target, JBP has merit. The protein shares little homology with other proteins in the Protein Data Bank, and it has a unique ligand, J-DNA containing telomeric stretches of double-stranded DNA, that does not occur in other eukaryotes. However, a preliminary high-throughput screen, focused on disrupting binding between JBP-1 and J-DNA, with a library of compounds consisting of all the major drug pharmacophoric groups has revealed no compounds of interest.

In parallel, we have studied the molecular recognition that underlies JBP-1 recognition of glycosylated DNA. In collaboration with D.P. Millar and D.A. Case, Department of Molecular Biology, we found that JBP-1 interacts with the J-containing DNA only when a critical conformation of the glucose within the major groove is established (Fig. 2).

Fig. 2. Surface rendering of a molecular dynamics snapshot of the critical 'edge-on' conformation of glucose (stick representation) within the major groove essential for recognition of J-DNA by JBP-1.

Publications

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.

Grover, R.K., Pond, S.J., Cui. Q., Subramanian, P., Case, D.A., Millar, D.P., Wentworth, P., Jr. O-Glycoside orientation is an essential aspect of base J recognition by the kinetoplastid DNA-binding protein JBP1. Angew. Chem. Int. Ed. 46:2839, 2007.

Nieva, J., Shafton, A., Altobell, L.J. III, Tripurenani, S., Lerner, R.A., Wentworth, P., Jr. Inflammatory aldehydes accelerate antibody light chain aggregation. Biochemistry, in press.

Witter, D., Wentworth, P., Jr. The antibody-catalyzed water-oxidation pathway from discovery to an emerging role in health and disease. Antioxid. Redox Signal., in press.

Xu, Y., Hixon, M.S., Yamamoto, N., McAllister, L.A., Wentworth, A.D., Wentworth, P., Jr., Janda, K.D. Antibody-catalyzed anaerobic destruction of methamphetamine. Proc. Natl. Acad. Sci. U. S. A. 104:3681, 2007.