Spotlight pubs.acs.org/crt
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ANTHRAX LETHAL TOXIN PACKS A DEADLY DOUBLE PUNCH
The symptoms of anthrax, a notorious and potentially deadly disease caused by Bacillus anthracis infection, can be triggered by exposure to the bacterial protein cluster lethal toxin (LT). Although it is clear that LT induces cardiovascular collapse and death, which is apparently related to the induction of hypoxia in susceptible tissues, there were hints that LT also interferes with cellular capacity to respond to hypoxia. The normal cellular response to hypoxia is the upregulated expression of proteins involved in anaerobic metabolism and survival. However, a team of researchers led by David M. Frucht observed that both mice and cells exposed to LT paradoxically showed reduced indications of anaerobic metabolism compared to levels that would normally occur in hypoxic organisms ((2014) J. Biol. Chem., 289, 4180−4190). The research team hypothesized that LT interfered with hypoxia-inducible factor 1 (HIF-1), a critical transcription factor that controls expression of hypoxia-response proteins. Ultimately, they discovered that LT inhibits translation of HIF-1α, a subunit of HIF-1, reducing basal levels of HIF-1α in normoxic cells and blocking the normal accumulation of HIF-1α in hypoxic cells. The team demonstrated that cells with upregulated levels of HIF-1α were more likely to survive LT treatment than cells containing basal, normoxic levels of HIF-1α. The authors conclude that anthrax pathology results from a synergistic combination of LT-induced hypoxia and inhibition of the homeostatic response to hypoxia. Heidi A. Dahlmann
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MICROFLUIDICS DEVICE ENABLES SENSITIVE, SPECIFIC PROTEIN DETECTION
Genomics technologies enabling the detection of genetic biomarkers have greatly facilitated the advancement of personalized medicine, but complementary methods for monitoring protein biomarkers are relatively undeveloped. Typical protein sensors involve affinity-based assays coupled with optical or electronic detection. However, the applicability of these methods can be limited because of expense, interference from the sample matrix, and difficulty in simultaneously optimizing the signal-to-noise ratio and limit of detection. Ronald W. Davis, Mehdi Javanmard, and co-workers recently developed a novel proteomic platform to circumvent these problems ((2014) Proc. Natl. Acad. Sci. U.S.A., 111, 2110− 2115). Protein abundance and activity can be measured in their microfluidic device, which is composed of a sample incubation chamber, a filtered channel, and a detection chamber containing an electrical impedance sensor. In the corresponding assay, the sample is introduced into the incubation chamber, where the target protein binds to immobilized antibodies. After an hour, the sample is rinsed out of the chamber, and then immobilized target protein is adsorbed onto antibody-coated magnetic beads. After rinsing, target-bound beads are funneled through the detection chamber, where they generate a signal by creating resistance between active electrodes. The authors found that their assay could detect target molecules at concentrations one to two orders of magnitude lower than those detectable by ELISA, a standard protein assay, while requiring only one-tenth of the sample volume. Heidi A. Dahlmann © 2014 American Chemical Society
ANTILEUKEMIC PARTHENOLIDE DERIVATIVES SYNTHESIZED VIA P450-MEDIATED C−H FUNCTIONALIZATION
Adapted from Kolev et al. (2014) ACS Chem. Biol., 9, 164−173. Copyright 2014 American Chemical Society.
Natural products are often important leads for pharmaceutical development, with the sesquiterpene lactone parthenolide being of interest because of its anticancer activity against acute myelogeneous leukemia (AML) primary and stem cells. Elaborating the parthenolide scaffold by selective functionalization of inactivated C−H bonds is difficult to achieve using currently available chemical methods, which prompted a research team led by Rudi Fasan to consider using oxidative P450 enzymes to obtain parthenolide derivatives for structure−activity relationship studies ((2014) ACS Chem. Biol., 9, 164−173). After generating a panel of P450 variants derived from the bacterial CYP102A1 enzyme, the research team was able to identify and optimize variants with high regioselectivity and catalytic efficiency for oxidizing either C9 or C14 of parthenolide. Although the C9- and C14-hydroxylated analogues were much less potent than the parent compound, their benzoylated derivatives were 2−4-fold more potent than parthenolide against two types of AML primary cells and retained good selectivity for AML rather than normal bone marrow cells. The authors note Published: April 21, 2014 455
dx.doi.org/10.1021/tx500106j | Chem. Res. Toxicol. 2014, 27, 455−456
Chemical Research in Toxicology
Spotlight
A new door for investigating human Pol ζ has just been opened by a research team led by Wei Yang ((2014) Proc. Natl. Acad. Sci. U.S.A., 8, 2954−2959). To enable purification, the group engineered a catalytically active form of Pol ζ with a Rev3 subunit that was about two-thirds the size of the wild-type protein. In pull-down assays with the mutant variant, the team also isolated two additional accessory subunits of Pol ζ, PolD2 and PolD3, which greatly enhanced the efficiency and processivity of Pol ζ. In a demonstration of the utility of their four-subunit Pol ζ (Pol ζ4) construct for biochemical studies, the team showed that Pol ζ4 was able to assist another TLS polymerase, Pol η, in bypassing a cisplatin-induced DNA cross-link. Heidi A. Dahlmann
that because the C9 and C14 postions were previously inaccessible by chemical manipulation the ability to engineer and screen P450 variants was very important for producing promising parthenolide derivatives. Heidi A. Dahlmann
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SILVER NANOPARTICLE TOXICITY GOES BEYOND SILVER ION TOXICITY
Adapted from Ivask et al. (2014) ACS Nano, 8, 374−386. Copyright 2014 American Chemical Society.
For nearly a century, humans have battled microbes with colloidal silver formulations containing silver nanoparticles (AgNPs). Although it is evident that dissolution of Ag+ ions from AgNPs contributes to AgNP toxicity, it is also known that NP size and coating can attenuate the biological effects of AgNPs, possibly because of the impact of these physicochemical properties on Ag+ dissolution and delivery. From these observations, one might assume that the toxicity of all AgNPs is mediated through the same mechanism, namely, the action of free Ag+ ions on their bacterial target. However, Hilary A. Godwin and co-workers have recently shown that this is not always the case ((2014) ACS Nano, 8, 374−386). The research team screened a library of about 4000 Escherichia coli mutants for their sensitivity to a series of AgNPs possessing various sizes and coatings. They found that only 35 strains were sensitive to each type of AgNP as well as AgNO3, a source of soluble Ag+, and they determined that these strains were deficient in genes related to ion transport, DNA damage and oxidative stress responses, and inner-membrane proteins. For the most part, each type of AgNP produced a unique pattern of response among the E. coli mutants, indicating diverse mechanisms of action. Furthermore, the researchers determined that Ag+ bioavailability and NP surface charge were correlated with NP toxicity. These results underscore the importance of fully characterizing NP physicochemical properties as well as considering multiple toxicological end points during future NP toxicity studies. Heidi A. Dahlmann
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ELUSIVE HUMAN TRANSLESION SYNTHESIS POLYMERASE PURIFIED Bulky DNA lesions, such as UV-induced dimers and chemotherapy drug-induced DNA cross-links, form strong barriers to normal DNA replication. However, translesion synthesis (TLS) polymerases, such as eukaryotic DNA polymerase ζ (Pol ζ), are able to bypass potentially cytotoxic DNA lesions. Pol ζ, composed of a catalytic and an accessory subunit (Rev3 and Rev7, respectively), is essential for mammalian cell proliferation and embryogenesis as well as TLS. Biochemical and structural studies of human Pol ζ have been hampered by difficulties in purifying the enzyme, which, with >3000 residues, is twice as large as its better-studied yeast homologue. 456
dx.doi.org/10.1021/tx500106j | Chem. Res. Toxicol. 2014, 27, 455−456
