LUPUS SERA--PROBES OF THE APOPTOTIC MECHANISM

白细胞介素下游底物的鉴定 1 β-转化酶（CE）家族，其聚集裂解 改变关键的细胞结构和功能， 细胞凋亡仍然是一个重大挑战。 广泛、长期 该提案的目标是定义蛋白水解途径， 细胞死亡，从而突出了潜在的治疗目标， 其中不适当的凋亡是突出的各种疾病 （包括恶性肿瘤、自身免疫和神经退行性疾病） 疾病）。 建议的具体目标是：（1）使用 识别抗原的狼疮自身抗体在 凋亡，以确定实际的分子切割;（2）以确定 负责切割每个分子的蛋白酶;（3） 阐明这些裂解发生的位点;以及（4） 确定产生的片段的生理功能， 切割，从而解决片段本身是否 重要的信号转换器。 自身抗体将 用于通过筛选人cDNA来鉴定底物 表达式库。 裂解位点将通过肽测定 测序研究和新的ICE样切割位点的鉴定将 用于形成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 不同纯化底物的切割效率 纯化的重组ICE样蛋白酶（包括ICE，ICE-2， ICE-3、Ich-1、CPP 32、Mch 2、Mch-3、FLICE/MACH和Ced-3）将 此外，还可以测定不同的细胞因子的抑制的IC 50值。 底物的一组四肽醛抑制剂， 凋亡提取物。 这些定量数据将表明， ICE样活性很可能是导致 凋亡细胞中的特定底物。 瞬时和稳定 转染编码通过以下步骤产生的片段的cDNA ICE样蛋白酶切割将用于解决 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.