Biological consequences of protein oxidation

蛋白质氧化的生物学后果

• 批准号: 6545293
• 负责人: EMILY B SHACTER
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6545293
• 关键词: B cell lymphoma; antioxidants; apoptosis; cell growth regulation; cell line; chemical structure function; cisplatin; cytotoxicity; disease /disorder model; etoposide; free radical oxygen; hydrogen peroxide; lipid metabolism; oxidation; oxidative stress; oxidizing agents; protein metabolism; western blottings

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.

总结：氧化剂如过氧化氢（H2 O2）涉及介导多种人类疾病，包括动脉粥样硬化、癌症和神经退行性疾病。 氧化剂通过对生物分子造成损害和改变细胞代谢而促进疾病过程。 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，重要的是要发现哪些蛋白质受到氧化应激的影响，它们被修饰的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地方是癌症化疗药物诱导的肿瘤细胞死亡。 据推测，细胞内的氧化剂（活性氧; ROS）中产生的癌症化疗药物治疗的细胞，这些是药物诱导的细胞凋亡过程的重要组成部分。 如果是这样，那么这些氧化剂可能通过修饰细胞活力所需的细胞内分子起作用。 我们特别感兴趣的是寻找可能被凋亡相关氧化剂修饰的特定蛋白质，因为这些蛋白质可能催化传播或抑制凋亡级联反应的反应。 我们的研究涉及使用人B淋巴瘤细胞作为肿瘤模型，并且我们采用了两种不同的癌症化疗药物，其有时被引用为通过促氧化机制起作用：VP-16和顺铂。 通过测定（1）蛋白质羰基（Western blot免疫测定法）、（2）蛋白质甲硫氨酸亚砜残基（氨基酸分析法）、（3）蛋白质巯基氧化（Western blot免疫测定法）来评估蛋白质氧化。 我们还测量了F2-异前列烷的形成作为脂质过氧化的标志物，以及使用氧化剂敏感染料DCFDA和DHR 123的细胞内ROS产生。 我们的研究结果表明，这两种药物诱导广泛的细胞凋亡的情况下，任何可检测到的蛋白质或脂质氧化，在细胞的胞浆和线粒体区室测量。 相反，H2 O2通过非凋亡途径杀死细胞，导致蛋白质和脂质氧化增加。 三种不同的抗氧化剂化合物（N-乙酰半胱氨酸，Tempol和MnTBAP）未能抑制药物诱导的细胞凋亡，同时抑制H2 O2诱导的细胞死亡。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的机制，不涉及氧化剂，不需要蛋白质氧化。 这一发现可能对改善化疗方案有重要的影响，因为它表明，通过在治疗方案中加入饮食抗氧化剂，可以安全地减少癌症化疗中氧化剂介导的副作用。