LUPUS SERA--PROBES OF THE APOPTOTIC MECHANISM

白介素酶下游底物的鉴定 1β转换酶(CE)家族，其聚集的裂解 改变关键的细胞结构和功能，从而产生 细胞凋亡仍然是一个重大挑战。广泛的、长期的 这项提案的目标是定义蛋白质分解途径，以 细胞死亡，从而突出了潜在的治疗靶点 多种疾病，其中不适当的凋亡是突出的 (包括恶性肿瘤、自身免疫性和神经退行性变 疾病)。该提案的具体目的是(1)利用 识别早期裂解抗原的狼疮自身抗体 细胞凋亡鉴定实际裂解的分子；(2)鉴定 负责裂解每个分子的蛋白酶(S)；(3) 阐明这些卵裂发生的位置；及(4) 确定由其产生的碎片的生理功能 切割，从而解决片段本身是否 重要的细胞凋亡信号转导系统。自身抗体会 用于筛选人cDNAs底物 表达式库。裂解位点将由多肽决定 测序研究和发现的新的类似ICE的切割位点将 被用来制造适当的四肽抑制剂作为工具 更有选择性地定义其他ICE样蛋白酶。催化剂 不同纯化底物的裂解效率 纯化的重组ICE样蛋白酶(包括ICE，ICE-2， ICE-3、ICH-1、CPP32、Mch2、MCH-3、FLICE/MACH和CED-3)将 被测定的IC50值也对不同的抑制作用 底物由一组四肽醛抑制剂在 凋亡性提取物。这一量化数据将表明哪些 冰样活动可能是导致分裂的原因。 凋亡细胞中的特定底物。暂态和稳定 编码所产生的片段的cDNA的转染 冰状蛋白水解酶将被用来解决 哺乳动物细胞中的这些片段。

The identification of downstream substrates for the interleukin 1beta-converting enzyme (CE) family, whose aggregated cleavage alters critical cell structures and functions, thereby generating apoptosis, remains a major challenge. The broad, long-term objectives of this proposal are to define the proteolytic routes to cell death, thereby highlighting potential therapeutic targets in a variety of diseases in which inappropriate apoptosis is prominent (including malignancy, autoimmunity, and neurodegenerative diseases). The specific aims of the proposal are (1) to use the lupus autoantibodies that recognize antigens cleaved early during apoptosis to identify the actual molecules cleaved; (2) to identify the protease(s) responsible for cleaving each molecule; (3) to elucidate the sites at which these cleavages occurs; and (4) to determine the physiologic functions of fragments generated by cleavage, thereby addressing whether the fragments themselves are important transducers of the apoptotix signal. Autoantibodies will be used to identify substrates by screening human cDNA expression libraries. Cleavage sites will be determined by peptide sequencing studies, and novel ICE-like cleavage sites identified will be used to fashion appropriate tetrapeptide inhibitors as tools to more selectively define the other ICE-like proteases. The catalytic efficiency of cleavage of the different purified substrates by purified recombinant ICE-like proteases (including ICE, ICE-2, ICE-3, Ich-1, CPP32, Mch2, Mch-3, FLICE/MACH and Ced-3) will be determined, as well the IC50 values for inhibition of different substrates by a panel of tetrapeptide aldehyde inhibitors in apoptotic extracts. This quantitative data will indicate which ICE-like activity(ies) are likely to be responsible for cleaving particular substrates in the apoptotic cell. Transient and stable transfections of cDNAs encoding the fragments generated by ICE-like protease cleavage will be used to address the effects of these fragments in mammalian cells.