Site-specific fragmentation of superoxide dismutase and DNA damage

我们已经报道了一种细胞质蛋白铜，锌-超氧化物歧化酶，在糖尿病和衰老的条件下经历糖基化、碎裂和失活。我们检测了糖化铜锌超氧化物歧化酶对DNA损伤的影响。我们发现，克隆的DNA与糖化的铜，锌-超氧化物歧化酶孵育后出现片段化。从细胞中分离的核仁DNA中也观察到了这种片段化。金属离子螯合剂和羟基自由基清除剂抑制DNA的断裂，提示糖化的铜锌超氧化物歧化酶和释放的铜离子参与了Fenton反应。我们还利用杆状病毒和昆虫细胞建立了人铜锌超氧化物歧化酶的表达系统，并产生了三种与肌萎缩侧索硬化症相关的突变酶(Gly41Asp，His43Arg，Gly85Arg)和野生型酶并进行了纯化。Gly85Arg突变体的活性与野生型相似，但Gly41Asp和His43Arg的活性降低了近一半。突变酶的结构不稳定性和过氧化氢的失活可能是导致ALS活性下降的原因之一，提示ALS可能是由突变酶的这种活性下降引起的。线粒体酶Mn-SOD由肿瘤坏死因子和白介素1诱导，清除细胞因子处理产生的活性氧。结果表明，转化生长因子-β处理不仅对锰超氧化物歧化酶有抑制作用，而且对铜、锌超氧化物歧化酶、过氧化氢酶、谷胱甘肽-S转移酶等抗氧化酶均有抑制作用。这些结果表明，TGB-β诱导的细胞凋亡是由抗氧化酶活性降低所触发的。

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.