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The Skaggs Institute
for Chemical Biology


Scientific Report 2008




Chemical Approaches to Disease

P. Wentworth, Jr., R.K. Grover, B.D. Song, M.M.R. Peram, J. Rogel, R.P. Troseth, D. Angrish, V. Dubrovskaya, A.D. Wentworth

Our interdisciplinary research focus 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. Currently, we are validating a new antileishmania drug target and increasing the scope of the role of inflammatory aldehydes in protein misfolding diseases.

The genomic DNA of kinetoplastid parasites contains a unique modified base, 5-(β-D-glucopyranosyloxymethyl)-2′-deoxyuridine or base J. Recently, we reported the first in-depth analysis of the molecular recognition between the O-linked glycoside component of deoxyribose J (dJ) in telomeric dJ-containing double-stranded DNA and J-binding protein 1 (JBP1) of Crithidia fasciculata. Comparison between the molecular dynamics snapshots and the free energy of binding of JBP1 to a panel of duplex oligonucleotides containing telomeric modified dJ revealed that JBP1 binding to dJ-containing oligonucleotides occurs preferentially when the β-D-glucopyranosyl moiety adopts a conformation within the major groove wherein the C-2 and C-3 hydroxyl groups of the glucoside make hydrogen-bond contacts to the nonbridging pro-R phosphoryl oxygen of the J-1 nucleotide phosphate group. If this orientation is perturbed even slightly, JBP1 binding affinity drops to the level of replacement of dJ by deoxythymidine.

In the past year, we have expanded this work and shown that this same edge-on conformation occurs in JBP1 in nonpathogenic Leishmania tarantolae. We also designed phosphorothioate probes to replace the J-1 residue in the duplex DNA. We hypothesized that replacement of the J-1 phosphoryl oxygen with sulfur would have a clear effect on the strength of the C-2 and C-3 hydrogen bonding. This hypothesis was proved by the finding that JBP1 does not bind to the phosphorothioate-containing duplex DNA as tightly as to normal duplex DNA.

In conclusion, the use of new phosphorothioate J-1 DNA as a chemical tool has validated our proposed binding hypothesis for JBP1 to J-DNA. This study further emphasizes the importance of our earlier proposed hypothesis that glucose conformation in the major groove of DNA is due to hydrogen bonds between the quasi-equatorial C-2 and C-3 hydroxyl groups of the sugar and the pro-R phosphoryl oxygen of the J-1 nucleotide. This understanding has clear ramifications for structure-based drug design of therapeutics against Leishmania parasites.

Recently, we discovered a process that we are studying in the context of several disease-related sporadic amyloidoses. We have shown that in vitro, certain inflammatory-derived lipidic aldehydes, when adducted to proamyloidogenic proteins in the proteins' native state, can induce misfolding and aggregation of the native protein sequences. This past year, we expanded our studies to aggregation of antibody light chains. In vivo, such aggregation leads to the systemic deposition of immunoglobulin light-chain domains in the form of either amyloid fibrils (AL-amyloidosis) or amorphous deposits (light-chain deposition disease), mainly in cardiac or renal tissue, and is a pathologic condition that is often fatal. Molecular factors that may contribute to the propensity of antibody light chains to aggregate in vivo, such as the protein primary structure or local environment, are intensive areas of study.

We have now shown that aggregation of human antibody κ (κ-MJM) and λ (λ-L155) light chains can be accelerated in vitro when they are incubated under physiologically relevant conditions in the presence of a panel of biologically relevant lipid-derived aldehydes: 4-hydroxynonenal, malondialdehyde, glyoxal, atheronal-A, and atheronal-B. Thioflavin-T and Congo red binding assays coupled with turbidity studies revealed that this aldehyde-induced aggregation can be associated with alteration of protein secondary structure to an increased β-sheet conformation. We found that the nature of the conformational change depends primarily on the lipidic aldehyde, not the protein sequence. Thus, the cholesterol 5,6-seco-sterols, atheronal-A and atheronal-B, caused amorphous aggregations that did not bind thioflavin-T or Congo red for both light chains, whereas 4-hydroxynonenal, malondialdehyde, and glyoxal induced aggregates that bound both thioflavin-T and Congo red. Transmission electron microscopy revealed that amyloid fibrils were formed during the aggregation of κ-MJM and λ-L155 light chains mediated by 4-hydroxynonenal (Fig. 1), whereas aggregates induced by atheronal-B were amorphous.
Fig. 1.Transmission electron microscopy image of fibrillar aggregates of antibody light chains induced by 4-hydroxynonenal in vitro.

Kinetic profiles of light-chain aggregation revealed clear differences between the aldehydes. Atheronal-A and atheronal-B caused a classic nucleated polymerization-type aggregation, with a lag phase (~150 hours) followed by a growth phase that plateaued, whereas 4-hydroxynonenal, malondialdehyde, and glyoxal triggered a seeded-type aggregation that has no lag phase. Studies of the accelerated aggregation of κ-MJM and λ -L155 induced by 4-hydroxynonenal revealed a clear dependence on the concentration of the aldehyde and a process that can be inhibited by the naturally occurring osmolyte trimethylamine N-oxide. On the basis of these data, we think that our recently discovered model of protein misfolding induced by inflammatory aldehydes may now extend to aggregation of antibody light chains.

Publications

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

Scheinost, J.C., Boldt, G.E., Wentworth, P., Jr. The chemical biology of protein misfolding. In: Encyclopedia of Chemical Biology. Wiley-VCH, 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.

Temperinini, 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-methanesulfonamide 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