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Scientific Report 2008


Chemistry




Chemistry, Biology, and Inflammatory Disease


P. Wentworth, Jr., D. Angrish, J. Dambacher, V. Dubrovskaya, R.K. Grover, J. Nieva, M. Puga, B.D. Song, M.M.R. Peram, J.K. 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. We are interested in uncovering new mechanisms of disease in major conditions such as atherosclerosis, neurodegenerative diseases, ischemia-reperfusion injury, macular degeneration, cancer, and infectious diseases.

Antibody-Catalyzed Water Oxidation Pathway

Our discovery that all antibody molecules can catalyze the reaction between singlet oxygen and water to give hydrogen peroxide is causing a revision of the idea that antibodies are only an adapter molecule within the immune system, linking recognition and killing of foreign pathogens. We are exploring both the chemical and biological aspects of this pathway, and new insights into how the pathway plays 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 (VHVL, CH1CL, VH, VL, CH1, CL) of the murine Fab 4C6. All of the domains can generate hydrogen peroxide when presented with singlet dioxygen, suggesting that the driving force is related to the immunoglobulin fold of the whole antibody.

INFLAMMATORY ALDEHDYES AND PROTEIN
MISFOLDING

We have shown that the inflammation-derived cholesterol seco-sterols atheronal-A and atheronal-B 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 his group, Department of Chemistry, we extended this model and showed that these cholesterol seco-sterols also trigger the misfolding of amyloid β-peptide1-40, leading to formation of fibrils similar to those observed in patients with Alzheimer's disease. Using mutated synthetic sequences of amyloid β-peptide1-40, we found that the accelerated aggregation of this protein only occurs when only lysine 16, not lysine 28 or the N-terminal amino group of aspartic acid 1, of the sequence is modified. More recently, in studies of inflammatory aldehyde—initiated misfolding of antibody light chains (Bence-Jones proteins), we found that different aldehydes can trigger different forms of aggregation in different proteins. Thus, we have shown that the cholesterol seco-sterols atheronal-A and atheronal-B accelerate an amorphous form of aggregation, whereas 4-hydroxynonenal induced an amyloid form of aggregation of both λand κlight chains (Fig. 1).


Fig. 1. Electron micrograph of fibrillar aggregation of antibody light chains induced by cholesterol seco-sterol and 4-hydroxynonenal (shown in white).

Epidemiologic and clinical evidence point to an increased risk of cancer when linked with chronic inflammation, in a process thought to involve the establishment of a local inflammatory microenvironment conducive to the development of neoplasia. However, because of the complex interrelationships between the 2 conditions, the precise molecular and cellular mechanisms that underpin this relationship remain largely unresolved.

We found that the inflammation-derived cholesterol 5,6-seco-sterol aldehydes atheronal-A and atheronal-B cause a loss of function of wild-type tumor suppressor protein p53, the so-called guardian of the genome, in a process that involves p53 misfolding and amyloidogenesis. Atheronal-A and atheronal-B, but not the aldehydes 4-hydroxynonenal and 4-hydroxyhexenal derived from polyunsaturated fatty acids, induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo red dye but cannot bind to a consensus DNA sequence (Fig. 2). Treatment of lung carcinoma cells expressing wild-type p53 with atheronal-A and atheronal-B leads to dysfunctional p53, as determined by analysis of extracted nuclear protein and transcription activation of p21.
Fig. 2. Optical microscopy images (100X) obtained with normal (upper) and cross-polarized (lower) light of aggregates generated by incubation of hexahistidine-tagged native p53 with atheronal-A and stained with Congo red.

Our results reveal a hitherto unknown chemical link between inflammation and cancer and expand the already pivotal role of p53 dysfunction in the risk for cancer. The increasing generality and specificity of aldehyde-initiated protein misfolding suggests 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 misfolding diseases.

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, thereby increasing drug resistance and 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 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. More recently, we discovered that low micromolar concentrations of the DNA intercalators daunorubicin and mitoxantrone disrupt the binding of JBP-1 with duplex DNA containing J-DNA. Modeling suggests that DNA binding of the intercalators leads to distortion, which leads to disruption of the edge-on conformation of the glucose within the major groove of the DNA.

Publications

Grover, R.K., Wentworth, P., Jr. Emerging therapies for kinetoplastid diseases. Prog. Infect. Dis., in press.

Nieva, J., Shafton, A., Altobell, L.J. III, Tripurenani, S., Rogel, J.K., Wentworth, A.D., Lerner, R.A., Wentworth, P., Jr. Inflammatory aldehydes accelerate antibody light chain amyloid and amorphous aggregation. Biochemistry 47:7695, 2008.

Scanlan, C.N., Ritchie, G.E., Baruah, K., Crispin, M.D., Harvey, D.J., Singer, B.B., Lucka, L., Wormald, M.R., Wentworth, P., Jr., Zitzmann, N., Rudd, P.M., Burton, D.R., Dwek, R.A. Inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, HIV-1 specific antibody. J. Mol. Biol. 372:16, 2007.

Scheinost, J.C., Boldt, G.E., Wentworth, P., Jr. Protein misfolding diseases. In: Encyclopedia of Chemical Biology, Wiley Blackwell, New York, in press.

Scheinost, J.C., Wang, H., Boldt, G.E., Offer, J., Wentworth, P., Jr. Cholesterol seco-sterol-induced aggregation of methylated amyloid-β peptides, insights into aldehyde-initiated fibrillization of amyloid-β . Angew. Chem. Int. Ed. 47:3919, 2008.

Temperini, C., Cecchi, A., Boyle, N.A., Scozzafava, A., Cabeza, J.E., Wentworth, P., Jr., Blackburn, G.M., Supuran, C.T . Carbonic anhydrase inhibitors. Interaction of 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methylsulfonamide with 12 mammalian isoforms: kinetic and x-ray crystallographic studies. Bioorg. Med. Chem. Lett. 18:999, 2008.

Wentworth, P., Jr., Witter, D. Antibody-catalyzed water-oxidation pathway. Pure Appl. Chem. 80:1849, 2008.

 

Paul Wentworth, Jr., Ph.D.
Professor

Anita Wentworth, Ph.D.
Assistant Professor



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