Biological consequences of protein oxidation

过氧化氢(H_2O_2)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。以前关于蛋白质氧化的研究包括将纯化的蛋白质暴露于氧化剂(如铁/抗坏血酸)源中，并测量损伤的程度和后果。最近，我们致力于研究化疗药物对肿瘤细胞蛋白质氧化的诱导作用。文献报道表明，化疗药物诱导细胞凋亡是由细胞内产生的氧化剂介导的。我们开始确定蛋白质是否在这一过程中被氧化，如果是的话，特定蛋白质的氧化修饰是否有助于细胞凋亡过程。在过去的一年里，人们进行了实验，以确定在化疗诱导的细胞凋亡和过氧化氢诱导的坏死过程中可能发生氧化的特定蛋白质。重点是确定可能被修饰的线粒体蛋白，因为线粒体功能是凋亡过程的核心。用药物VP-16诱导细胞凋亡，并通过测定蛋白质羰基(Western印迹实验)和总蛋氨酸亚砜(与美国国立卫生研究院NHLBI的研究人员合作)来评估蛋白质氧化。到目前为止，我们还没有发现VP-16诱导的细胞凋亡过程中蛋白质氧化增加的证据。抗氧化剂对VP-16诱导的细胞死亡无明显抑制作用。我们的结果提供了证据，证明细胞凋亡不一定涉及氧化应激，也不需要蛋白质氧化。这一发现意义重大，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中不可或缺的一部分。这项工作目前正准备在同行评议的期刊上发表。

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.