Spotlight Probing for Cysteine Sulfenic Acid Endogenous and exogenous reactive oxygen species (ROS) are known causes of cellular damage. When excessive ROS production overwhelms cellular defense mechanisms, oxidative stress results. The study of oxidative stress requires accurate measurement of the chemical consequences of ROS exposure, such as the one proposed by Takanishi et al. [(2007) Biochemistry 46, 14275]. Yap1 is a transcription factor responsible for regulating oxidant-dependent transcription in Saccharomyces cerevisiae. Cys598 of the Yap1 c-terminal cysteine-rich domain (cCRD) detects oxidant damage through disulfide bond formation with cysteine sulfenic acids, such as those generated in peroxiredoxins upon oxidant exposure. Takanishi et al. expressed
DNA Break? Call RNF8! Cellular damage that is severe enough to cause a double strand break in DNA requires an immediate and efficient repair response. Cells exposed to ionizing radiation suffer such damage and respond by building a complex of proteins, the ionizing radiation-induced focus (IRIF), at the damage site. IRIF formation is initiated by the ATM kinase, which phosphorylates histone H2AX, forming γ-H2AX. γ-H2AX triggers the accumulation of additional proteins, including MDC1, NBS1, 53BP1, and BRCA1. Now, in nearly simultaneous reports from four research groups encompassing six independent laboratories, we learn of the role of a key protein, RNF8, in the assembly and function of the IRIF [Kolas et al. (2007) Science 318, 1637; Mailand et al. (2007) Cell 131, 887; Huen et al. (2007) Cell 131, 901; and Wang and Elledge (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 20759. The four groups took different approaches to their discovery of RNF8. Kolas et al. screened an siRNA library in search of proteins that are required for the incorporation of 53BP1 into the IRIF. Wang and Elledge used the fact that the E2 ubiquitin-conjugating enzyme, UBC13, is involved in IRIF formation, so they searched for E3 ubiquitin ligases that interact with UBC13. Both Huen et al. and Mailand et al. capitalized on the knowledge that FHA- and RING domaincontaining proteins are frequently involved in DNA repair, so they focused on proteins bearing these characteristics. In every case, the single protein identified was RNF8. Extensive studies on the role of RNF8 in IRIF formation led to the following conclusions. Upon initiation of a double strand break, formation of γ-H2AX leads to the rapid accumulation of MDC1, NBS1, and RNF8 to the damage site. MDC1 is required for the binding of the other two 274
Vol. 21,
No. 2,
•
CHEMICAL RESEARCH IN TOXICOLOGY
His6-tagged cCRD in Escherichia coli and demonstrated that H2O2 exposure led to complex formation between the cCRD and other cellular proteins. Dimedone, which reacts with cysteine sulfenic acid, prevented complex formation. When cell lysates were passed over a nickel affinity column, the complexes bound to the column. Following elution of nonadherent proteins, the researchers used dithiothreitol to break the disulfide bonds and then elute the complexed proteins. This method allowed the identification of six E. coli proteins known to exhibit cysteine sulfenic acid formation upon oxidation plus nine other proteins known to have reactive cysteine residues. This interesting method should be readily adaptable to the study of oxidative stress in mammalian cells as well. • Carol A. Rouzer
proteins, but NBS1 and RNF8 bind independently of one another. An intact FHA domain in RNF8 is required for its interaction with MDC1, which contains four TQXF motifs to which the FHA domain binds tightly following phosphorylation of the threonine residue by ATM. Once bound, RNF8 works together with UBC13 to promote K63 ubiquitylation of histones. Both H2 and H2AX histones are substrates for ubiquitylation, which requires an intact RNF8 RING domain. Rap80 is an adaptor molecule bearing two UIM domains that mediate binding to K63 polyubiquitylated proteins. Rap80 also bears an AIR (abraxas interacting region) domain, through which it binds abraxas, which then serves as an adaptor to bind BRCA1. 53BP1 binding to the IRIF does not depend on Rap80, but it is dependent on RNF8, UBC13, and ubiquitylation at the damage site. Thus, following the rapid aggregation of MDC1, NBS1 and RNF8 at the IRIF, 53BP1, and BRCA1 subsequently appear after histone ubiquitylation catalyzed by RNF8. However, it is not clear if their arrival depends upon histone ubiquitinylation or some other ubiquitinylation substrate. These conclusions were reached through the application of multiple methods in the six laboratories, and each
Reprinted from Mailand et al. (2007) Cell 131, 887, with permission from Elsevier. Published online 02/18/2008
•
DOI: 10.1021/tx8000048 $40.75 © 2008 American Chemical Society
Spotlight laboratory contributed additional unique insights into IRIF formation and function. For full appreciation, it is well worth checking out all four of these articles. • Carol A. Rouzer
Do Not Cross Your DNA While the cancer-causing ability of many chemicals is firmly linked to their mutagenic activity, the DNA lesions causing the mutations are not yet fully understood. Research on chemically induced DNA damage has largely been focused on binary carcinogen-DNA adducts produced when DNA and the chemical of interest are reacted in a test tube. However, DNA lesions in cells are not created in such an isolated environment. One large class of DNA adducts that has been almost completely ignored by mutagenesis research includes DNA–protein and DNApeptide cross-links. Many human carcinogens are known to cause DNApeptide and DNA–protein cross-links (which may be proteolytically processed into DNA-peptide cross-links) in cells or in vitro. Despite this, there is a dearth of research into the effects of these lesions. In a new study, Minko et al. [(2008) Mutat. Res. 637, 161] provide important insight into the mutagenic properties of DNA-peptide cross-links. The authors modeled cross-links formed after acrolein exposure and found that mutagenic effects of these modifications in mammalian cells were dependent on the site of attachment to DNA. They discovered that peptides attached at the N6 position of deoxyadenosine in the major DNA groove were not significantly mutagenic. In contrast, cross-links at the N2 position of deoxyguanosine in the minor DNA groove caused almost 10 times more mutations than binary DNA adducts. The authors’ findings have opened an avenue of investigation into a previously overlooked form of DNA damage that is expected to contribute considerably to the mutagenicity of other bifunctional DNA-damaging carcinogens. • Elizabeth Bartley and Anatoly Zhitkovich
Downfall of Lumiracoxib Selective inhibitors of cyclooxygenase-2 (COX-2) were proposed as the ideal substitutes for nonsteroidal antiinflammatory drugs (NSAIDS) for the treatment of pain and inflammation. The selective inhibition of the COX-2 isoform, which is predominantly associated with inflammation, while leaving the constitutively expressed COX-1 unaffected, leads to fewer gastrointestinal side effects than are observed with the nonselective NSAIDS. However, clinical use of COX-2 inhibitors has revealed unexpected toxicities leading to the removal of rofecoxib (Vioxx), eterocoxib (Bextra), and, most recently, lumiracoxib (Prexige) from global markets.
Published online 02/18/2008 • DOI: 10.1021/tx8000048 © 2008 American Chemical Society
$40.75
Rofecoxib and eterocoxib cause cardiovascular and epidermal toxicities, respectively, effects that are believed to be mechanism-based. In contrast, lumiracoxib is hepatotoxic, and now, Li et al. [(2007) Drug Metab. Dispos. published online Nov. 12, DOI:10.1124/dmd.107.019018] propose a metabolism-based mechanism for lumiracoxib’s toxicity, based on its similarity in structure to diclofenac. Incubation of lumiracoxib with human liver microsomes or human hepatocytes in the presence of N-acetylcysteine (NAC) yields two products, 3′-NAC-4′-hydroxy lumiracoxib and 4′-hydroxy-6′-NAC-desfluoro lumiracoxib. In both cases, the reactions are attributed to cytochrome P450 2C9, and both involve the generation of a reactive quinone imine intermediate. Thus, as in the case of diclofenac, a likely mechanism for lumiracoxib hepatotoxicity is P450-catalyzed bioactivation to an electrophilic intermediate that damages surrounding tissue. • Carol A. Rouzer
Searching for 5-Methylcytosine Evidence is growing for the role of DNA base methylation in the epigenetic control of chromatin structure and function. Aberrant methylation patterns have been associated with various disease states, such as cancer and inflammation, and 5-methycytosine (5MedC) residues, present at CpG sequences, are particularly prone to mutagenesis. Consequently, the ability to identify 5MedC residues in DNA is key to fully understanding the role of DNA methylation in the mechanism of toxic species that act in the nucleus. Bareyt and Carell [(2008) Angew. Chem. Int. Ed. 120, 187] address this issue in work aimed at finding conditions that will selectively react with 5MedC but no other DNA bases. Because of reactivity at the C5-C6 double bond, the redox potential of 5MedC is slightly lower than that of dC or dT, suggesting that selective oxidation should be possible. Reaction of a test methylated DNA strand with V2O5 at pH 3-5 showed that, indeed, 5MedC reacted whereas dT and dC did not. However, reaction with dG also occurred, attributed to direct electron abstraction, rather than electrophilic attack on a double bond. The latter reaction could be suppressed by the addition of LiBr and/or by the removal of oxygen. However, a search for more “user friendly” reaction conditions led to the identification of NaIO4/LiBr at pH 5 and 40 °C as ideal for the highly efficient and selective oxidation of 5MedC residues. Coupled with hot piperidine-induced strand breaks at the modified bases, this reaction was used to identify the 5MedC residues in a 40 base pair fragment from the promoter of p16, an important gene in the regulation of basal cell carcinoma proliferation. These results form the framework for the development of a new, robust assay for the detection of 5Me dC in complex DNA samples. • Carol A. Rouzer TX8000048
Vol. 21,
No. 2,
•
CHEMICAL RESEARCH IN TOXICOLOGY
275
