PEROXIREDOXIN THAT FORMS AN INTRAMOLECULAR DISULFIDE

过氧化物氧还蛋白（Prx）家族的过氧化物酶含有一个Cys残基，其前面是NH 2-末端区域中的保守序列。一种新型的哺乳动物Prx，命名为PrxV，现在已经被确定为数据库搜索的结果，这个保守的含Cys序列。162个氨基酸的PrxV与先前鉴定的哺乳动物Prx酶仅具有约10%的序列同一性，并且除了对应于保守Cys的Cys残基（Cys 48）之外，还在位置73和152处含有Cys残基。分析突变的人PrxV蛋白，其中这三个半胱氨酸残基中的每一个被单独替换为丝氨酸表明，巯基的Cys 48是由过氧化物氧化的网站，和氧化的Cys 48与巯基的Cys 152反应，形成分子内二硫键。因此，PrxV的氧化中间体与其他Prx酶的氧化中间体不同，其他Prx酶形成分子间二硫化物或次磺酸中间体。PrxV形成的二硫化物被硫氧还蛋白还原，但不被谷氧还蛋白或谷胱甘肽还原。因此，缺乏Cys 48或Cys 152的PrxV突变体没有表现出可检测的硫氧还蛋白依赖性过氧化物酶活性，而Cy 73突变对活性没有影响。免疫印迹分析表明，PrxV广泛表达于大鼠组织和培养的哺乳动物细胞，并定位于细胞内的细胞质，线粒体和过氧化物酶体。在NIH 3 T3细胞中，野生型蛋白质的瞬时表达而非Cy 48突变体的瞬时表达抑制了肿瘤坏死因子-α诱导的H2 O2积累和c-Jun NH 2-末端激酶的激活，从而证明了PrxV在体内的过氧化物酶功能。

Peroxidases of the peroxiredoxin (Prx) family contain a Cys residue that is preceded by a conserved sequence in the NH2-terminal region. A new type of mammalian Prx, designated PrxV, has now been identified as the result of a database search with this conserved Cys-containing sequence. The 162-amino acid PrxV shares only ~10% sequence identity with previously identified mammalian Prx enzymes and contains Cys residues at positions 73 and 152 in addition to that (Cys48) corresponding to the conserved Cys. Analysis of mutant human PrxV proteins in which each of these three Cys residues was individually replaced with serine suggested that the sulfhydryl group of Cys48is the site of oxidation by peroxides, and that oxidized Cys48 reacts with the sulfhydryl group of Cys152 to form an intramolecular disulfide linkage. The oxidized intermediate of PrxV is thus distinct from those of other Prx enzymes, which form either an intermolecular disulfide or a sulfenic acid intermediate. The disulfide formed by PrxV is reduced by thioredoxin, but not by glutaredoxin or glutathione. Thus, PrxV mutants lacking Cys48 or Cys152 showed no detectable thioredoxin-dependent peroxidase activity, whereas mutation of Cy73 had no effect on activity. Immunoblot analysis revealed that PrxV is widely expressed in rat tissues and cultured mammalian cells, and is localized intracellularly to cytosol, mitochondria, and peroxisomes. The peroxidase function of PrxV in vivo was demonstrated by the observations that transient expression of the wild-type protein, but not that of the Cy48 mutant, in NIH 3T3 cells inhibited H2O2 accumulation and activation of c-Jun NH2-terminal kinase induced by tumor necrosis factor-alpha.