AUTOANTIGEN CLEAVAGE DURING APOPTOSIS AND NECROSIS

凋亡和坏死是两种基本的细胞死亡途径， 是对生理激活剂、物理 创伤或环境毒素和化学物质。 这些细胞模式 死亡与广泛的人类病理状况有关， 包括系统性自身免疫、癌症、艾滋病和神经退行性疾病 紊乱 新的证据表明半胱氨酸的激活 ICE/CED-3家族的蛋白酶是免疫系统中的核心机制。 凋亡的执行。虽然导致细胞坏死的机制 仍然是模糊的，最近的研究还指出，激活蛋白酶 是细胞死亡过程中的关键事件 然而，这些的性质 蛋白酶和它们的底物在很大程度上仍是未知的。 使用人 抗核自身抗体作为探针，我们已经证明， 凋亡和坏死涉及选择性的，但不同的是， 一组特定的核蛋白的不同的蛋白水解裂解 自身抗原 有人提出，不同的蛋白水解机制， 是细胞凋亡和坏死的基础， 这些机制应有助于明确区分 在这些细胞死亡过程之间。 广泛的长期目标 这项研究是为了剖析和区分与 这些机制。 提出了三个具体目标，以测试我们的 假说. (1)使用一组抗核自身抗体， 抗原靶标是充分表征的核蛋白，作为探针， 定义和区分底物蛋白水解相关的模式 Fas介导的细胞凋亡和由毒素诱导的坏死 异生汞。 (2)表征涉及的蛋白水解活性 在两种细胞死亡过程中特异性自身抗原的裂解中。 细胞凋亡和坏死的无细胞系统将用于这些研究。 分析自身抗原切割抑制特征的研究， 为了确定ICE/CED-3蛋白酶的激活是否在 坏死 纯化蛋白酶切割特异性抗原的能力 并对特异性切割位点的鉴定进行研究。 (3)为了利用在小鼠中观察到的自身抗原切割的差异， 细胞凋亡和坏死的单克隆抗体的发展 其特异性地与与细胞凋亡相关的表位反应， 或坏死细胞死亡。 预计这些研究将 有助于提高我们对细胞死亡的理解， 条款和建立额外的标准， 区分凋亡和坏死。

Apoptosis and necrosis are two fundamental cell death pathways that may be the end result of response to physiologic activators, physical trauma, or environmental toxins and chemicals. These modes of cell death are associated with a wide range of human pathological conditions, including systemic autoimmunity, cancer, AIDS, and neurodegenerative disorders. Emerging evidence indicates that activation of cysteine proteases of the ICE/CED-3 family is a central mechanism in the execution of apoptosis. Although the mechanisms driving cell necrosis are still obscure, recent studies also point to activation of proteases as a key event in this cell death process. However, the nature of these proteases and their substrates remains largely unknown. Using human antinuclear autoantibodies as probes, we have demonstrated that both apoptosis and necrosis involve the selective, but distinctively different, proteolytic cleavage of a specific set of nuclear protein autoantigens. It is proposed that different proteolytic mechanisms underlie the execution of apoptosis and necrosis, and that dissecting these mechanisms should be helpful for establishing a clear distinction between these cell death processes. The broad, long term objectives of this research is to dissect and differentiate events associated with these mechanisms. Three specific aims are proposed to test our hypothesis. (1) To use a panel of antinuclear autoantibodies where the antigen targets are well characterized nuclear proteins, as probes to define and differentiate patterns of substrate proteolysis associated with Fas-mediated apoptosis and with necrosis induced by the toxic xenobiotic mercury. (2) To characterize proteolytic activities involved in the cleavage of specific autoantigens in both cell death processes. Cell free systems of apoptosis and necrosis will be used in these studies to analyze the inhibition profile of autoantigen cleavage and to determine whether activation of ICE/CED-3 proteases occurs during necrosis. The ability of purified proteases to cleave specific antigens and the identify of specific cleavage sites will be also investigated. (3) To exploit the differences in autoantigen cleavage observed in apoptosis and necrosis for the development of monoclonal antibodies which react specifically with epitopes associated with either apoptotic or necrotic cell death. It is anticipated that these studies should contribute to enhancing our understanding of cell death in molecular terms and establishing additional criteria that would help to differentiate between apoptosis and necrosis.