CYSTEINE PROTEASES IN APOPTOSIS AND CANCER

细胞凋亡性死亡对正常发育和维持至关重要 正常组织的动态平衡。越来越多的证据表明，监管失调 细胞凋亡可能会导致几种人类疾病，包括癌症， 自身免疫性疾病和退行性神经元疾病，如 阿尔茨海默氏症和帕金森氏症。新出现的证据表明 蛋白水解酶的激活可能是导致 执行细胞凋亡。与线虫中CED-3半胱氨酸蛋白酶不同 是细胞凋亡的主要执行者，最近的观察表明 高等真核生物的细胞凋亡可能涉及多个半胱氨酸 与CED-3和ICE相关的蛋白酶。这些蛋白水解酶可能是 一种可扩增的凋亡蛋白酶级联类似于 补体激活。半胱氨酸蛋白酶的数量仍在增加， 现就其激活机制、在细胞凋亡中的作用及相关研究进展作一综述。 细胞凋亡的底物在很大程度上仍不清楚。要隔离和 我们开发了一种新的半胱氨酸蛋白酶的聚合酶链式反应技术 富含编码高度保守的五肽的DNA序列 CED-3/ICE样半胱氨酸凋亡中存在QACRG和GSWFI/GSWYI 蛋白酶。我们克隆了几种类似CED-3/ICE的蛋白水解酶 可能是脊椎动物细胞凋亡分子机制的组成部分。 因此，建议过度表达全长，并截断和 这些半胱氨酸蛋白酶在果翅夜蛾中的突变衍生物 (Sf9)细胞和在大肠杆菌中的表达。这将使大量的这些 用于功能和结构研究的蛋白质。哺乳动物细胞凋亡 本实验室克隆的半胱氨酸蛋白酶，将对其进行鉴定。他们的 将分析基因的组织特异性和时间性表达，以及 存在选择性剪接的异构体。潜在的底物和 这些蛋白水解酶的抑制剂将使用生化和 分子生物学技术。预计这些研究将 有助于阐明这一重要因素的激活机制 一类蛋白酶。这将加强努力确定他们的 相关的内源性底物，并设计特定的药物 规范他们的活动。除了将获得的价值之外 从了解细胞凋亡在发育和发育中的分子机制 动态平衡，拟议的研究也将产生重要的知识 可用于治疗许多人类疾病，如 癌症和许多其他退行性疾病。

Apoptotic cell death is essential for normal development and maintenance of normal tissue homeostasis. There is growing evidence that dysregulation of apoptosis may lead to several human diseases including cancer, autoimmune disorders and degenerative neuronal diseases such as Alzheimer's and Parkinson's diseases. Emerging evidence suggests that protease activation might be the central mechanism leading to the execution of apoptosis. Unlike in C. elegans where CED-3 cysteine protease is the major executioner of apoptosis, recent observations suggest that execution of apoptosis in higher eukaryotes may involve multiple cysteine proteases related to CED-3 and ICE. These proteases, might be components of an amplifiable apoptotic protease cascade similar to the cascade of complement activation. The number of cysteine proteases is still growing, however their mechanism of activation, role in apoptosis and relevant apoptotic substrates are still largely unknown. To isolate and characterize novel cysteine proteases we developed a PCR technique to enrich for DNA sequences which encode the highly conserved pentapeptides QACRG and GSWFI/GSWYI present in CED-3/ICE-like apoptotic cysteine proteases. We were able to clone several CED-3/ICE-like proteases that could be components of the molecular mechanism of vertebrate apoptosis. Consequently, it is proposed to overexpress full length, and truncated and mutated derivatives of these cysteine proteases in Spodoptera frugiperda (Sf9) cells and in E. coli. This will avail large quantities of these proteins for functional and structural studies. Mammalian apoptotic cysteine proteases cloned in our laboratory, will be characterized. Their genes will be analyzed for tissue specific and temporal expression and the presence of alternatively spliced isoforms. Potential substrates and inhibitors of these proteases will be identified using biochemical and molecular biology techniques. It is anticipated that these studies will contribute to elucidation of the mechanism of activation of this important class of proteases. This will enhance the efforts to identify their relevant endogenous substrates and to design specific drugs that will regulate their activity. In addition to the value which will be gained from understanding the molecular mechanism of apoptosis in development and homeostasis, the proposed studies will also generate significant knowledge that could be applied in the treatment of many human diseases such as cancer and many other degenerative diseases.