Dopamine-induced apoptosis of PC12 cells

细胞中的多巴胺（DA）降解产生 H2 O2，它可以进一步还原为羟基自由基（OH）， 铁的存在。DA衍生的OH引起的细胞损伤 已被广泛研究，与发病机制有关， 帕金森氏症。我们建立了一个程序， 具有H2 O2敏感的Cys（或Secys）残基的蛋白质， 在PC 12细胞中DA衍生的H2 O2对蛋白质的氧化。的 该方法是基于这样的事实，即在pH 6.5，H2 O2和 生物素偶联碘乙酰胺（BIAM）选择性， 竞争性地与低pKa值的Cys残基反应。减少 在BIAM标记中，由于细胞先前暴露于DA， 随后用辣根过氧化物酶缀合的 在SDS-凝胶上分离后的链霉亲和素。使用该过程， PC 12细胞中的许多蛋白质显示具有Cys残基 对DA衍生的H2 O2氧化敏感。显著 靶点包括ERP 72和ERP 60，这两种蛋白质 二硫键异构酶家族和肌酸激酶。磷脂酶 C-γ 1和硫氧还蛋白还原酶， PC 12细胞也被DA衍生的H2 O2氧化。通过使用 [14 C]碘乙酰胺代替BIAM，甘油醛3-磷酸 脱氢酶被鉴定为靶标。的体外研究 纯化的酶表明，必需残基，Cys 283的 3-磷酸甘油醛肌酸激酶 硫氧还蛋白还原酶的Secys 498特异性地 被H2 O2氧化。虽然确定的目标只代表一个 被DA衍生的H2 O2修饰的蛋白质部分， 它们功能的损害以前与 细胞死亡。因此，我们建议，蛋白质的氧化与 DA-衍生的H2 O2的反应性半胱氨酸还原可能在很大程度上 负责DA诱导的神经元细胞凋亡， 帕金森病发病机制中的核心角色。

Dopamine (DA) degradation in cells generates H2O2, which can be further reduced to hydroxyl radicals (OH ) in the presence of iron. Cellular damages inflicted by DA-derived OH have been studied extensively in relation to the pathogenesis of Parkinsonian's disease. We established a procedure that detects proteins with H2O2-sensitive Cys (or Secys) residues and studied protein oxidation by DA-derived H2O2 in PC12 cells. The procedure is based on the fact that at pH 6.5, H2O2 and biotin-conjugated iodoacetamide (BIAM) selectively and competiviely react with Cys residues with low pKa value. Decrease in BIAM-labeling due to prior exposure of cells to DA was followed by blot analysis with horseradish peroxidase-conjugated streptavidin after separation on SDS-gels. Using the procedure, a number of proteins in PC12 cells were shown to have Cys residues that are sensitive to oxidation by DA-derived H2O2. Notable targets include ERP72 and ERP60, two members of protein disulfide isomerase family and creatine kinase. Phospholipase C-gamma1 and thioredoxin reductase, two minor components of PC12 cells, were also oxidized by DA-derived H2O2. By using [14C]iodoacetamide instead of BIAM, glyceraldehyde 3-phosphate dehydrogenase was identified as a target. In vitro studies with purified enzymes suggest that the essential residues, Cys283 of creatine kinase, Cys151 of glyceraldehyde 3-phosphate dehydrogenase, Secys498 of thioredixin reductase are specifically oxidized by H2O2. Although the identified targets represent only a portion of proteins that are modified by DA-derived H2O2, impairment of their functions have previously been closely related to cell death. We propose therefore that oxidation of proteins with reactive cysteine reisdues by DA-derived H2O2 might be largely responsible for the DA-induced apoptosis of neuronal cells and a central player in the pathogenesis of Parkinson's disease.