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Crosslinking of DNA and proteins induced by protein hydroperoxides.

ABSTRACT: Exposure of DNA to several proteins peroxidized by radiation-generated hydroxyl free radicals resulted in formation of crosslinks between the macromolecules, detected by retardation and broadening of DNA bands in agarose gels. This technique proved suitable for the study of crosslinking of DNA with peroxidized BSA, insulin, apotransferrin and alpha casein, but not with several other proteins, including histones. The crosslinking depended on the presence of intact hydroperoxide groups on the protein, on their number, and on the duration of the interaction with DNA. All DNA samples tested, pBR322, pGEM, lambda/HindIII and pUC18, formed crosslinks with the peroxidized BSA. Sodium chloride and formate prevented the crosslinking if present during incubation of the peroxidized protein and DNA, but had no effect once the crosslinks had formed. The gel shift of the crosslinked DNA was reversed by proteolysis, indicating that the DNA mobility change was due to attachment of protein and that the crosslinking did not induce DNA strand breaks. The metal chelators Desferal and neocuproine reduced the extent of the crosslinking, but did not prevent it. Scavengers of free radicals did not inhibit the crosslink formation. The DNA-protein complex was not disrupted by vigorous agitation, by filtration or by non-ionic detergents. These observations show that the crosslinking of DNA with proteins mediated by protein hydroperoxides is spontaneous and probably covalent, and that it may be assisted by transition metals. It is suggested that formation of such crosslinks in living organisms could account for some of the well-documented forms of biological damage induced by reactive oxygen species-induced oxidative stress.

SUBMITTER: Gebicki S

PROVIDER: S-EPMC1220096 | biostudies-other | 1999 Mar

REPOSITORIES: biostudies-other

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Project description:Loss of innervation is a key driver of age associated muscle atrophy and weakness (sarcopenia). Our laboratory has previously shown that denervation induced atrophy is associated with the generation of mitochondrial hydroperoxides and lipid mediators produced downstream of cPLA2 and 12/15 lipoxygenase (12/15-LOX). To define the pathological impact of lipid hydroperoxides generated in denervation-induced atrophy in vivo, we treated mice with liproxstatin-1, a lipid hydroperoxide scavenger. We treated adult male mice with 5 mg/kg liproxstain-1 or vehicle one day prior to sciatic nerve transection and daily for 7 days post-denervation before tissue analysis. Liproxstatin-1 treatment protected gastrocnemius mass and fiber cross sectional area (∼40% less atrophy post-denervation in treated versus untreated mice). Mitochondrial hydroperoxide generation was reduced 80% in vitro and by over 65% in vivo by liproxstatin-1 treatment in denervated permeabilized muscle fibers and decreased the content of 4-HNE by ∼25% post-denervation. Lipidomic analysis revealed detectable levels of 25 oxylipins in denervated gastrocnemius muscle and significantly increased levels for eight oxylipins that are generated by metabolism of fatty acids through 12/15-LOX. Liproxstatin-1 treatment reduced the level of three of the eight denervation-induced oxylipins, specifically 15-HEPE, 13-HOTrE and 17-HDOHE. Denervation elevated protein degradation rates in muscle and treatment with liproxstatin-1 reduced rates of protein breakdown in denervated muscle. In contrast, protein synthesis rates were unchanged by denervation. Targeted proteomics revealed a number of proteins with altered expression after denervation but no effect of liproxstain-1. Transcriptomic analysis revealed 203 differentially expressed genes in denervated muscle from vehicle or liproxstatin-1 treated mice, including ER stress, nitric oxide signaling, Gαi signaling, glucocorticoid receptor signaling, and other pathways. Overall, these data suggest lipid hydroperoxides and oxylipins are key drivers of increased protein breakdown and muscle loss associated with denervation induced atrophy and a potential target for sarcopenia intervention.

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Crosslinking of DNA and proteins induced by protein hydroperoxides.

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