Spotlight 8-OxoG and Transcription A common lesion resulting from oxidative DNA damage is 8-oxoguanine (8-OG), which is repaired primarily by the base excision repair (BER) pathway initiated by 8-OG glycosylase (Ogg1). Some evidence exists that 8-OG may also be repaired by the transcription-coupled repair (TCR) pathway, although this pathway usually targets bulkier lesions. Failure to repair 8-OG in the coding strand of DNA may result in transcriptional mutagenesis (TM), in which miscoding during transcription produces a mutant mRNA and subsequently an aberrant protein. Now, Saxowsky et al. [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 18877] report a new method to monitor TM in intact mammalian cells. An expression plasmid encoding H-ras carries either wild-type guanine or 8-OG at codon 61. If not repaired, TM at the 8-OG site leads to a constitutively active Q61K
The MARTX Toxin Link The MARTX toxin of Vibrio cholerae is an example of a bacterial toxin that acts by direct modification of Actin subunits. The result is disassembly of stress fibers and cell rounding, leading to failure of phagocytes to clear invading bacteria from the intestine. Prior studies have shown that the ACD domain of MARTX cross-links actin monomers, but the exact nature of the modification was not known until Kudryashov et al. carried out their recently reported findings [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 18537]. Limited proteolysis of dimers formed by ACD treatment of actin monomers showed that the cross-link was formed between peptides 48 and 68 on one monomer and 227 and 375 on the second monomer. X-ray diffraction of crystals formed from cross-linked dimers and gelsolin segment 1 or gelsolin segment 1 plus DNaseI localized the cross-link to residues K50 and E270. Further support for this conclusion came from mass spectrometric studies of yeast actin bearing a cysteine residue at position 59. Following treatment with ACD and limited trypsinization, the Cys59 residue provided a point of attachment for a biotin affinity tag, allowing easy isolation of the cross-linked peptide. The resulting mass spectral data unequivocally verified an amide link between K50 and E270. Finally, Kudryashov et al. showed that actin monomers bearing mutations of the K50 or E270 residues could not be cross-linked by ACD either in vitro or when expressed in HeLa cells. The locations of K50 and E270 in actin’s hydrophobic loop and DNaseI-binding loop, respectively, distance them from the sites of interaction of many proteins that bind actin monomers. This explains why proteins such as thymosin β4 and profilin do not interfere with ACD-dependent crossPublished online 02/16/2009 • DOI: 10.1021/tx800486e © 2009 American Chemical Society
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mutant protein and an increase in phosphorylation of the downstream kinase ERK. Transfection of the plasmid into wild-type MEF cells or MEF cells null for Ogg1 and/or for the TCR protein Cockayne syndrome B (Csb) demonstrated that repair of 8-OG required Ogg1 but not Csb. TM, as detected by increased ERK phosphorylation, occurred in ogg1-/-csb+/+ cells and ogg-1-/-csb-/- cells but not in ogg-1+/+csb-/- or wild-type cells. The findings strongly support a role for BER but not TCR in the repair of 8-OG in this intact mammalian cell system. They also suggest that aberrant proteins produced by TM may contribute to the generation of a malignant phenotype if the target protein, as in the case of H-ras, is critical for the control of cell division and differentiation. •Carol A. Rouzer
link formation. The conformations of the hydrophobic and DNasI-binding loops render K50 and E270 readily available for ACD action. The resulting cross-link does not directly impinge on the actin polymerization site; however, it does force the actin subunits into a conformation that is not amenable to normal polymerization and filament formation.
This figure was kindly provided by Emil Reisler.
Kudryashov et al. point out that the ATP-dependent formation of a cross-link between the carboxyl group and the primary amino group of nonterminal amino acids on two separate peptides is a unique feature of ACD action. Vol. 22,
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Spotlight Thus, the MARTX toxin represents an expansion of bacterial toxic mechanisms. •Carol A. Rouzer
Ricin’s Way In The plant cytotoxin ricin enters cells via multiple pathways following binding to surface galactosyl residues. After retrograde transport to the ER, ricin is reduced to its toxic A chain (RTA) and sugar-binding B chain (RTB). RTA then leaves the ER via the mechanism used by the ER-associated protein degradation (ERAD) pathway. Normally, this route would lead to proteasomal degradation, so the means by which RTA evades this fate is of considerable interest. Spooner et al. [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 17408] now show that interaction with the Hsc70 chaperone is critical to RTA’s survival.
Reproduced with permission from Spooner et al. [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 17408]. Copyright 2008 National Academy of Sciences.
Deoxyspergualin, which alters Hsc70s ATPase activity, protected HeLa cells from ricin’s toxicity, and Hsc70, in combination with cochaperone Hsp40 and ATP, reactivated RTA following heat denaturation. In contrast, geldanamycin and radicicol, which inhibit Hsp90s ATPase activity, sensitized HeLa cells to ricin, and transient overexpression of Hsc70-Hsp90 organizing protein (Hop), which transfers proteins from Hsc70 to Hsp90, protected cells from ricin toxicity. These initial results suggested that Hsc70 prevents RTA aggregation and promotes renaturation of the protein, while Hsp90 facilitates RTA degradation. Further studies showed that overexpression of BCL2-associated athanogene protein (BAG-1) or the C terminus of Hsp70 interacting protein (CHIP) protected cells from ricin cytotoxicity. Both of these proteins facilitate proteasomal degradation of Hsc70- and/or Hsp90-associated proteins. In contrast, overexpression of BAG-2 or Hsc70-interacting protein (Hip), which interferes with BAG-1 and stabilizes Hsc70-client protein complexes, respectively, increased HeLa cell sensitivity to ricin. Spooner et al. suggest that Hsc70 and its cochaperones determine the fate of RTA once it leaves 242
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the ER and propose that one role of Hsc70 is to uncouple ERAD substrates from the final destructive steps. Hsc70bound RTA, under the influence of Hsp90, Hop, BAG-1, and CHIP, will be degraded, whereas interaction with BAG-2 and Hip will result in stabilization. Active RTA in the cytosol damages ribosomes via depurination, resulting in failure of protein synthesis and, ultimately, cell death. •Carol A. Rouzer
Redox Control of mRNA Levels Ceruloplasmin (Cp), a well-known plasma copper transport protein, also plays a role in iron homeostasis. Studies of human patients lacking Cp and Cp knockout mice indicate iron overload in liver and brain, suggesting that Cp is important for the regulation of iron transport and storage. Because iron plays a key role in the generation of reactive oxygen species, Tapryal et al. [(2008) J. Biol. Chem., published online November 18, DOI: 10.1074/jbc.M804079200] hypothesized that Cp expression may be regulated under conditions of oxidative stress. Cp is synthesized in hepatocytes for secretion into the circulation and in astroglial cells, where it exists as a glycophosphatidylinositol-linked membrane protein. Tapryal et al. showed that exposure to H2O2 resulted in decreased levels of Cp protein in HepG2 human hepatocellular carcinoma cells and in human U373MG and rat C6 glial cells in culture. Similar results were obtained when cells were treated with antimycin A or rotenone, which promote endogenous H2O2 formation through blockade of mitochondrial electron transport. The decreased Cp protein levels were associated with a similar decrease in mRNA levels, which because of the slow natural turnover rate of Cp mRNA suggested an increase in the rate of mRNA degradation. Direct studies of mRNA turnover confirmed this hypothesis. By constructing an expression vector in which the 3′-UTR of the Cp mRNA was fused with the gene for chloramphenical acetyl transferase, Tapryal et al. were able to show that it is the 3′-UTR region that conveys mRNA instability in the presence of H2O2. Further studies using gelshift assays showed that a protein complex readily formed on the Cp 3′-UTR in the presence of cytosolic extracts of HepG2 cells. However, complex formation was impeded in the case of cytosol from cells pretreated with H2O2. Together, the results suggest that H2O2 and possibly similar reactive oxygen species interfere with Cp protein synthesis by promoting rapid degradation of the Cp mRNA. This effect appears to be mediated through regulation of complex formation at the 3′-UTR, although the exact mechanism by which this occurs is as yet undefined. The effects of oxidants on protein expression mediated through transcriptional regulation of antioxidant response elementcontaining genes are now well-known. The findings of Tapryal et al. may form the basis for additional regulatory mechanisms acting downstream of mRNA transcription. •Carol A. Rouzer TX800486E Published online 02/16/2009 •
DOI: 10.1021/tx800486e $40.75 © 2009 American Chemical Society
