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The Skaggs Institute
for Chemical Biology
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.
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| 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.
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