Biological consequences of protein oxidation

蛋白质氧化的生物学后果

• 批准号: 6433551
• 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/6433551
• 关键词: carbohydrate metabolism; carbonyl compound; cytotoxicity; fibrinogen; glycoproteins; human tissue; hydrogen peroxide; lipid metabolism; oncoproteins; oxidation; oxidative stress; protease inhibitor; protein metabolism; proteolysis; western blottings

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. Previous studies on protein oxidation involved exposing purified proteins to a source of oxidants (e.g., iron/ascorbate) and measuring the extent and consequences of the damage. More recently, we have directed our efforts towards studying induction of protein oxidation in tumor cells exposed to chemotherapy drugs. Reports in the literature suggest that induction of apoptosis by chemotherapeutic agents is mediated by oxidants generated within the cells. We set out to determine whether proteins become oxidized in this process and, if so, whether oxidative modification of specific proteins contributes to the apoptotic process. Over the past year, experiments were carried out to identify specific proteins which might undergo oxidation during chemotherapy-induced apoptosis and H2O2-induced necrosis. Emphasis was placed on identifying mitochondrial proteins that might become modified since mitochondrial function is so central to the apoptotic process. Apoptosis was induced with the drug VP-16 and protein oxidation was assessed by measuring protein carbonyls (Western blot assay) and total methionine sulfoxide (in collaboration with investigators at the NHLBI, NIH). To date, we have found no evidence of increased protein oxidation during VP-16-induced apoptosis. There was also no induction of lipid peroxidation, and antioxidant compounds were ineffective at inhibiting VP-16-induced cell death. Our results provide evidence that apoptosis does not necessarily involve oxidative stress and does not require protein oxidation. This finding is significant because it challenges the current dogma which suggests that formation of intracellular oxidants is an integral part of the apoptotic process. The work is currently being prepared for publication in a peer-reviewed journal.

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。 先前关于蛋白质氧化的研究涉及将纯化的蛋白质暴露于氧化剂源（例如，铁/抗坏血酸盐）并测量损伤的程度和后果。 最近，我们致力于研究暴露于化疗药物的肿瘤细胞中蛋白质氧化的诱导。 文献中的报告表明，化疗剂诱导细胞凋亡是由细胞内产生的氧化剂介导的。 我们着手确定蛋白质是否在这个过程中被氧化，如果是这样，特定蛋白质的氧化修饰是否有助于凋亡过程。 在过去的一年中，进行了实验，以确定特定的蛋白质，可能会发生氧化过程中化疗诱导的细胞凋亡和H2 O2诱导的坏死。 重点放在确定线粒体蛋白，可能成为修改，因为线粒体功能是如此核心的凋亡过程。用药物VP-16诱导细胞凋亡，并通过测量蛋白质羰基（蛋白质印迹法）和总甲硫氨酸亚砜（与NHLBI，NIH的研究人员合作）评估蛋白质氧化。 迄今为止，我们尚未发现VP-16诱导的细胞凋亡过程中蛋白质氧化增加的证据。 也没有诱导脂质过氧化，抗氧化化合物在抑制VP-16诱导的细胞死亡是无效的。 我们的研究结果提供的证据表明，细胞凋亡并不一定涉及氧化应激，并不需要蛋白质氧化。 这一发现是重要的，因为它挑战了目前的教条，即细胞内氧化剂的形成是凋亡过程的一个组成部分。 这项工作目前正在准备发表在一份同行评审的杂志上。