Oxygen is a double-edge sword for cells: It is necessary for life, but at the same time it is the source of damaging free radicals that attack DNA, proteins and cell membranes. Reactive oxygen species are known to directly damage DNA, but a new study published today in Chemistry and Biology suggests that they also harm genes via the oxidation of proteins that cling to DNA. Shana Kelley and colleagues from Boston College in Massachusetts show that singlet oxygen reacts with DNA-bound proteins to form amino acid peroxides, which then proceed to chemically cleave the DNA sugar-phosphate backbone, introducing single-strand breaks. Since cellular DNA is invariably coated with proteins, the production and activity of amino acid peroxides could be a major source of the DNA damage that is associated with aging (see ARF related news story), which remains the biggest risk factor for Alzheimer disease and other neurodegenerative diseases.
To compound the ravages of aging, old brains may lose the ability to repel the assault by oxygen and time, says another new report from Diego Ruano and colleagues in Seville, Spain. In their paper, which appeared online on June 17 in Neurobiology of Aging, they show that hippocampi from aged rats display defects in the pathways that normally handle damaged, unfolded proteins. These age-related alterations could set up older brains for any one of several neurodegenerative protein aggregation diseases, they conclude.
To look at the role of oxidized amino acids in DNA damage, Kelley’s graduate students and co-first authors Erin Prestwich and Marc Roy tested DNA-binding tripeptides of the sequence lys-X-lys for their ability to cleave plasmid DNA in vitro in the presence of singlet oxygen. When the middle residue was cysteine, histidine, tyrosine or tryptophan, the peptides caused significant strand breaks, corresponding to their ability to form peroxides with oxygen. The ability of different peptides to mediate DNA cleavage was dependent on the formation and stability of peroxides on the different side chains, with tryptophan (W) being the most effective. The mechanism of cleavage was different from the alkaline-labile base damage typical of direct oxygen attack on DNA, and involved a cut in the sugar-phosphate backbone. In fact, the presence of the W-containing tripeptide protected the DNA from direct base damage by oxygen, leading the authors to characterize tryptophan as a “molecular ‘double-edged sword’ that can both suppress and induce DNA damage.”
These experiments were all carried out in vitro on plasmid or oligonucleotide DNA, but Kelley’s group has published evidence that protein-mediated DNA damage probably occurs in vivo, as well. In the April 22 Angewandte Chemie, first author Lisa Wittenhagen and colleagues conjugated a tryptophan-containing version of the DNA-binding Tat peptide from HIV with a photoactivatable dye that generates singlet oxygen. With this complex, they were able to bring together in close proximity cellular DNA, a tryptophan residue, and reactive oxygen. When the authors introduced the complex into HeLa cells, light induced cell death, but only when the tryptophan-containing Tat was used—when glycine was substituted for the tryptophan, light had no effect on cell viability. “We are working on follow-up studies that will characterize the chemical products of the DNA damage caused by these agents. In addition, we plan to examine cell lines that lack repair factors that typically take care of this type of damage naturally, which would accurately mimic aged or diseased cells,” Kelley said.
Moving from damage to defense, the second paper investigated the response of brain to misfolded proteins in young and aged rats. Accumulation of misfolded proteins in the endoplasmic reticulum triggers a set of compensatory changes, inducing upregulation of chaperone proteins, attenuation of protein translation, and degradation of proteins by the proteasome. These changes constitute the unfolded protein response (UPR), and first author M. Paz Gavilan and his colleagues show that the response does not work so well in the hippocampus of old rats. Gavilan found that, compared to young (4- to 6-month-old) rats, aged (23- to 26-month-old), rats had lower levels of three of the four chaperone proteins measured, and had higher levels of ubiquitinated proteins. To measure the UPR under stress, the researchers injected the proteasome inhibitor lactacystin directly into the hippocampus of young or aged rats. They saw increased ubiquitinated protein levels after 24 hours in young rats, which decreased back to normal after four days. Older rats showed greater increases, and failed to upregulate two chaperones, Grp78 and PDI, that are considered central to the UPR. At the same time, markers of apoptosis increased—an effect not seen in the young animals. According to the authors, the results “reveal, for the first time in vivo, a dysfunction in the ability of aged rats to solve the stress conditions induced by protein accumulation.” Similar reductions in the Grp78 chaperone (see ARF related news story) and proteasome function (Keller et al., 2000) have been observed in AD, and the new work serves to further illuminate the intriguing links among aging, protein mishandling, and neurodegenerative disorders.—Pat McCaffrey
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- Keller JN, Hanni KB, Markesbery WR. Impaired proteasome function in Alzheimer's disease. J Neurochem. 2000 Jul;75(1):436-9. PubMed.
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- Paz Gavilán M, Vela J, Castaño A, Ramos B, del Río JC, Vitorica J, Ruano D. Cellular environment facilitates protein accumulation in aged rat hippocampus. Neurobiol Aging. 2006 Jul;27(7):973-82. PubMed.
- Wittenhagen P, Kronborg G, Weis N, Nielsen H, Obel N, Pedersen SS, Eugen-Olsen J. The plasma level of soluble urokinase receptor is elevated in patients with Streptococcus pneumoniae bacteraemia and predicts mortality. Clin Microbiol Infect. 2004 May;10(5):409-15. PubMed.
- Prestwich EG, Roy MD, Rego J, Kelley SO. Oxidative DNA strand scission induced by peptides. Chem Biol. 2005 Jun;12(6):695-701. PubMed.