Site-specific fragmentation of superoxide dismutase and DNA damage

我们已经报道了细胞质蛋白Cu，Zn-SOD，在糖尿病和衰老条件下经历糖化，片段化和失活。我们研究了糖基化Cu，Zn-SOD对DNA损伤的影响。结果表明，用糖基化Cu，Zn-SOD处理克隆DNA时，克隆DNA发生了断裂，从细胞中分离的核DNA也发生了断裂。金属离子螯合剂和羟自由基清除剂对Cu，Zn-SOD的DNA断裂有抑制作用，表明Cu，Zn-SOD的糖基化和Cu^<2+>释放参与了芬顿反应（Gly 41 Asp，His 43 Arg，Gly 85 Arg）以及野生型酶，并将它们纯化。Gly 85 Arg突变体酶活力与野生型酶相近，但Gly 41 Asp和His 43 Arg酶活力下降近一半，结构不稳定和过氧化氢失活可能是导致突变体酶活力下降的原因之一。提示ALS可能是由这种突变酶活性降低引起的。1并清除由细胞因子处理产生的活性氧。结果表明，TGF-β不仅抑制Mn-SOD，而且抑制Cu，Zn-SOD，过氧化氢酶，谷胱甘肽-S-转移酶等抗氧化酶。这些结果表明，TGF-β诱导的细胞凋亡是由抗氧化酶活性的降低引发的。

We have reported that a cytoplasmic protein Cu, Zn-SOD,undergoes glycation, fragmentation, and inactivation under diabetic conditions and aging. We have examined effects of glycated Cu, Zn-SOD on DNA damage. We found that cloned DNA was fragmented by incubation with glycated Cu, Zn-SOD.This fragmentation was also observed in nulcear DNA isolated from cells. Metal ion chelators and hydorxyl radical scavengers inhibited the DNA fragmentation, suggesting the involvement of the Fenton reaction mediated by glycated Cu, Zn-SOD and released Cu^<2+> in this reaction.We also established human Cu, Zn-SOD expression system using baculovirus and insect cells, and produced three mutant enzymes (Gly41Asp, His43Arg, Gly85Arg) related to amyotrophic lateral sclerosis (ALS) as well as wild-type enzyme and purified them. Gly85Arg mutant exhibited similar activity to the wild-type enzyme, but activities of Gly41Asp and His43Arg were decreased to nearly a half of it. Structural unstability and inactivation of mutant enzymes by hydrogen peroxide may be a cause of the decreased activities, suggesting that ALS may be induced by this decreased activity in mutant enzymes.A mitochondrial enzyme Mn-SOD is induced by TNF and IL-1 and scavenges reactive oxygen species produced by treatment with cytokines. Our results show that not only Mn-SOD bur also some antioxidative enzymes including Cu, Zn-SOD,catalase, glutathione-S-transferase, are suppressed by treatment with TGF-beta. These results suggest that TGB-beta-induced apoptosis is triggered by the reduction of antioxidative enyzme activities.