Biological consequences of protein oxidation

摘要：过氧化氢(H2O2)等氧化剂与多种人类疾病有关，包括动脉粥样硬化、癌症和神经退行性疾病。氧化剂通过破坏生物分子和改变细胞新陈代谢而导致疾病进程。氧化损伤的关键目标是结构蛋白和酶。为了了解氧化应激如何导致疾病，重要的是要了解哪些蛋白质会受到氧化应激的影响，它们被修饰到什么程度，以及修饰的功能后果。蛋白质氧化可能发挥重要生理作用的一个领域是癌症化疗药物诱导的肿瘤细胞死亡。有人假设，细胞内的氧化剂(活性氧种；ROS)是在接受癌症化疗药物治疗的细胞中产生的，这些是药物诱导细胞凋亡过程中的一个重要组成部分。如果是这样，那么这些氧化剂很可能通过改变细胞存活所需的细胞内分子来发挥作用。我们特别感兴趣的是寻找可能被与凋亡相关的氧化剂修饰的特定蛋白质，因为这些蛋白质可能会催化促进或抑制凋亡级联反应的反应。我们的研究涉及使用人类B淋巴瘤细胞作为肿瘤模型，我们使用了两种不同的癌症化疗药物，有时被认为是通过促氧化机制发挥作用的：VP-16和顺铂。蛋白质氧化通过蛋白免疫印迹法检测(1)蛋白质羰基，(2)氨基酸分析测定蛋白质蛋氨酸亚砜残基，(3)蛋白质巯基氧化通过蛋白质印迹免疫法测定。我们还使用氧化剂敏感染料DCFDA和DHR 123测量了F2-异前列腺素的形成作为脂质过氧化的标志，以及细胞内ROS的产生。我们的结果表明，这两种药物在没有检测到任何可检测到的蛋白质或脂肪氧化的情况下，都能诱导广泛的细胞凋亡，这在细胞的胞浆和线粒体中都可以测量到。相比之下，过氧化氢通过非凋亡途径杀死细胞，导致蛋白质和脂肪氧化增加。三种不同的抗氧化剂化合物(N-乙酰半胱氨酸、坦波尔和MnTBAP)在抑制过氧化氢诱导的细胞死亡的同时，未能抑制药物诱导的细胞凋亡。我们的结论是，至少在B淋巴瘤细胞中，药物诱导的细胞凋亡发生在一种不涉及氧化剂和不需要蛋白质氧化的机制上。这一发现可能对改进化疗方案具有重要的影响，因为它表明，通过在治疗方案中添加饮食抗氧化剂，癌症化疗的氧化剂介导的副作用可能会安全地减少。这项工作正在进行审查，将发表在同行评议的杂志上。

Summary: Oxidants such as hydrogen peroxide (H2O2) are implicated in mediating a wide array of human diseases including atherosclerosis, cancer, and neurodegenerative diseases. Oxidants contribute to disease processes by causing damage to biomolecules and altering cellular metabolism. Key among the targets for oxidative damage are structural proteins and enzymes. In order to understand how oxidative stress can cause disease, it is important to discover which proteins become affected by oxidative stress, to what degree they are modified, and the functional consequences of the modifications. One place where protein oxidation could play a significant physiological role would be in tumor cells induced to die by cancer chemotherapy drugs. It has been hypothesized that intracellular oxidants (reactive oxygen species; ROS) are generated in cells treated with cancer chemotherapy drugs and that these are an essential component of the drug-induced apoptotic process. If so, then these oxidants likely act by modifying intracellular molecules that are required for cell viability. We were particularly interested in finding specific proteins that might be modified by apoptosis-associated oxidants as these are likely to catalyze the reactions that propagate or inhibit the apoptotic cascade. Our studies have involved use of human B lymphoma cells as a tumor model and we employed two different cancer chemotherapy drugs which have sometimes been cited as working through a pro-oxidant mechanism: VP-16 and cisplatin. Protein oxidation was assessed by measuring (1) protein carbonyl groups by Western blot immunoassay, (2) protein methionine sulfoxide residues by amino acid analysis, (3) protein sulfhydryl oxidation by Western blot immunoassay. We also measured formation of F2-isoprostanes as a marker of lipid peroxidation, and intracellular ROS production using the oxidant-sensitive dyes DCFDA and DHR 123. Our results show that both drugs induce extensive apoptosis in the absence of any detectable protein or lipid oxidation, measured in both the cytosolic and mitochondrial compartments of the cell. In contrast, H2O2, which kills the cells by non-apoptotic pathways, causes increases in both protein and lipid oxidation. Three different antioxidant compounds (N-acetyl cysteine, Tempol, and MnTBAP) failed to inhibit drug-induced apoptosis while inhibiting H2O2-induced cell death. We conclude that, at least in B lymphoma cells, drug-induced apoptosis occurs using a mechanism that does not involve oxidants and does not require protein oxidation. This finding may have important ramifications for improving chemotherapy protocols because it suggests that oxidant-mediated side-effects from cancer chemotherapy might safely be diminished by addition of dietary antioxidants to the treatment protocols.The work is under review for publication in a peer-reviewed journal.