Spotlight ROS-Regulated Kinases Reactive oxygen species (ROS) play a role in numerous toxic mechanisms. Exposure to ROS triggers a sophisticated series of responses regulated by numerous signaling pathways. Preliminary evidence suggests that protein tyrosine kinases (PTKs) may be regulated by ROS, but studies carried out in vivo have not clearly discerned if this regulation is direct or indirect. To address this question, Kemble and Sun [(2009) Proc. Natl. Acad. Sci. U.S.A. 106, 500] have investigated the effects of the redox environment on purified PTKs in vitro. Incubation of the Src kinase in the presence of dithiothreitol (DTT) resulted in a 13-fold stimulation of activity that was reversed by exposure to H2O2. Mutation studies demonstrated that DTT-mediated activation was dependent on Cys-277. Oxidative inactivation of Src was accompanied by dimerization of the protein, which resulted from disulfide bond
formation between the Cys-277 residues of two Src molecules. Cys-277 is located in the small N-terminal lobe of the catalytic domain known as the Gly loop of Src. Kemble and Sun discovered that only eight of >90 PTKs have a cysteine at this position. One of these, FGFR1, exhibited DTT-dependent activation that was eliminated by mutation of its Gly loop cysteine residue. Similarly, the Csk kinase, which lacks a Gly loop cysteine, was not activated by DTT, but when a cysteine was introduced at the appropriate site by mutation, the enzyme became sensitive to DTT stimulation. These results suggest a mechanism by which a small number of PTKs may be modulated by ROS, thereby contributing to the cellular response to oxidative stress. • Carol A. Rouzer
DNA Damage Detection Nucleotide excision repair (NER) is the primary pathway by which bulky DNA lesions are excised and replaced. In bacteria, NER is initiated by three proteins, UvrA, UvrB, and UvrC. A UvrA-UvrB complex scans DNA, searching for abnormal bases. Once a damage site is recognized, UvrA “loads” UvrB onto the site and then dissociates. UvrB recruits UvrC, which incises the DNA on either side of the lesion. The interaction between these proteins is crucial to their ability to recognize and bind to the site of DNA damage. Pakotiprapha et al. [(2009) J. Biol Chem., published online March 13, DOI: 10.1074/jbc.M900571200] have explored the nature of the UvrA-UvrB interaction and use their results to propose a model for DNA lesion recognition. On the basis of prior data that defined the UvrA-UvrB interaction domains, Pakotiprapha et al. expressed each domain and then crystallized them together. This approach allowed them to ascertain the structure of the bound complex. Both proteins exhibited considerably more order in the bound crystal structure than in the crystal structures of the isolated proteins, suggesting that the protein domains have a high degree of flexibility until they interact with each other. The crystal structure also revealed that the primary interactions between the two proteins were polar, consisting of hydrogen bonds (direct and water-mediated) and ionic bonds. They confirmed the importance of these interactions through mutation studies. These revealed that three out of four UvrA mutations tested completely abolished UvrA-UvrB binding. Of five UvrB mutations, two completely eliminated and two reduced binding, whereas one had no effect. Thus, all of the proposed interactions that they tested except for one contributed to the UvrA-UvrB interaction. Published online 06/15/2009 • DOI: 10.1021/tx9001513 © 2009 American Chemical Society
$40.75
Figure kindly provided by David Jeruzalmi. Modeled UvrA·UvrB complex based on superposition of the corresponding domains onto the experimentally determined structure. Note that the proposed DNA binding path on UvrA (red cylinder) and the crystallographically established DNA binding site on UvrB are aligned.
Pakotiprapha et al. used these results combined with molecular mass determinations to propose a model for lesion recognition by the UvrA-UvrB complex. The model is in agreement with other data concerning the way that these proteins interact with DNA and provides the basis for further experiments to define these critical structures. • Carol A. Rouzer Vol. 22,
No. 6, •
CHEMICAL RESEARCH IN TOXICOLOGY 967
Spotlight Cyp Levels and Dioxin Toxicity Fate of Thimerosal The aryl hydrocarbon receptor (AHR) is known to most toxicologists by its ability to mediate increased expression of cytochromes P450 in response to xenobiotic polycyclic aromatic hydrocarbons (PAHs). This adaptive response leads to the metabolism and clearance of PAHs. However, the AHR is also believed to contribute to the toxicity of some pollutants such as dioxin (2,3,7,8-tetrachlorodibenzo-pdioxin), which are particularly difficult to clear from the body. Finally, studies with AHR knockout mice have led to the conclusion that the AHR plays a developmental role, particularly with regard to the vasculature. Upon ligand binding, the AHR disengages from associated chaperone proteins, translocates to the nucleus, and dimerizes with the AHR nuclear translocator (ARNT). The resultant heterodimeric transcription factor binds to dioxin responsive enhancers (DREs) to regulate the transcription of target genes. The P450s Cyp1a1 and Cyp1a2 are strongly induced by AHR activation and have long been used as biomarkers of PAH exposure. However, the exact role of these enzymes in the various aspects of AHR function is not entirely clear. Now, Nukaya et al. [(2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4923] shed new light on this important question. A highly conserved cluster of DRE sequences (DREC) is located between the genes for Cyp1a1 and Cyp1a2. Nukaya et al. found that homozygous knockout of this DREC eliminated and markedly reduced dioxin-mediated increased expression of Cyp1a1 and Cyp1a2, respectively. DREC-/- mice exhibited normal hepatic vascular development, indicating that induction of these major P450 genes is not essential for the developmental function of the AHR. When exposed to a single toxic dose of dioxin, DREC-/mice exhibited greater increases in plasma alanine aminotransferase levels and more intense inflammatory changes in the liver than wild-type mice. In contrast, the livers of wildtype mice were notable for more severe hydropic changes. The two groups of mice did not differ with regard to hepatic lipid accumulation. Further studies explored the effects of deletion of the individual P450 genes. The response of Cyp1a1-/- or Cyp1a2-/- mice to dioxin exposure was similar to that of DREC-/- mice with regard to aminotransferase levels and hepatic inflammation. However, Cyp1a1-/- mice exhibited hydropic changes similar to those of wildtype mice, while Cyp1a2-/- mice resembled the DREC-/mice with regard to this histopathologic marker. Together, the results suggest that induction of Cyp1a1 and Cyp1a2 expression by AHR activation plays a protective role with regard to some aspects of the hepatotoxicity of dioxin. This finding is contrary to the simpler models of toxicity in which P450 induction primarily promotes dioxin hepatotoxicity. However, the reduction in hydropic changes observed with Cyp1a2 knockout supports a toxic function for P450 gene induction in some cases. • Carol A. Rouzer
968
Vol. 22,
No. 6, •
CHEMICAL RESEARCH IN TOXICOLOGY
In 1999, the U.S. Food and Drug Administration, the American Academy of Pediatrics, and the Centers for Disease Control and Prevention issued a recommendation that thimerosal, a mercury-containing preservative, be removed from vaccines. The recommendation was based on a risk assessment that showed that vaccination of infants might lead to mercury exposure in excess of safety levels set by the Environmental Protection Agency. In 2001, ARC Research, an autism research and advocacy group, suggested that thimerosal in vaccines could be a cause of autism. This putative connection has been refuted by available clinical evidence, and thimerosal has been removed from most vaccines in the United States and all vaccines in Europe, but controversy about vaccine safety continues.
Reproduced with permission from Tru¨mpler et al. [(2009) Metallomics 1, 87]. Copyright 2009 Royal Society of Chemistry.
Despite its prominent place in the media, the mechanisms of thimerosal toxicity are not fully understood. In aqueous solution, it undergoes hydrolysis to ethylmercury, which is believed to be the toxic species; yet, the exact fate of ethylmercury in vivo is not known. Tru¨mpler et al. [(2009) Metalomics 1, 87] now propose to use a combination of liquid chromatography/inductively coupled plasma mass spectrometry (LC/ICP-MS) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS) to explore the reaction of thimerosal with human serum albumin (HSA) and β-lactoglobulin A (β-LGA). Tru¨mpler et al. incubated HSA or β-LGA with thimerosal under physiologic conditions and used LC/ICP-MS to detect the presence of 200Hg and 202Hg in the samples. Results confirmed the hydrolysis of thimerosal in the absence of protein, but in the presence of protein, all detectable mercury became protein associated. LC/ToF-MS confirmed the presence of one ethylmercury adduct on HSA and β-LGA, which was attributed to the presence of a single free cysteine in both. Following trypsinization of β-LGA, LC/ ToF-MS analysis of the peptides indicated the presence of the ethylmercury adduct on the single free cysteine. The results confirm the reactivity of thimerosal-derived ethylmercury with cysteine sulfhydryls and outline a powerful method to detect and identify these adducts in proteins. Application of these methods to determine the fate of ethylmercury from thimerosal in vivo is of considerable interest for future research. • Carol A. Rouzer TX9001513
Published online 06/15/2009 •
DOI: 10.1021/tx9001513 $40.75 © 2009 American Chemical Society
