CYSTEINE PROTEASES IN APOPTOSIS AND CANCER

凋亡性细胞死亡对于正常发育和维持至关重要 正常组织的自我平衡越来越多的证据表明， 细胞凋亡可能导致几种人类疾病， 自身免疫性疾病和变性神经元疾病， 阿尔茨海默氏症和帕金森氏症。新出现的证据表明 蛋白酶激活可能是导致 凋亡的执行。不像C。其中CED-3半胱氨酸蛋白酶 是细胞凋亡的主要执行者，最近的观察表明， 高等真核生物中细胞凋亡的执行可能涉及多个半胱氨酸 与CED-3和ICE相关的蛋白酶。这些蛋白酶可能是 一个类似于级联的凋亡蛋白酶级联， 补体激活半胱氨酸蛋白酶的数量仍在增长， 然而，它们激活机制、在细胞凋亡中的作用和相关的 细胞凋亡底物仍然是未知的。分离和 表征新型半胱氨酸蛋白酶，我们开发了PCR技术， 富集编码高度保守的五肽的DNA序列 QACRG和GSWFI/GSWYI存在于CED-3/ICE样凋亡半胱氨酸中 蛋白酶我们能够克隆几种CED-3/ICE样蛋白酶， 可能是脊椎动物细胞凋亡的分子机制的组成部分。 因此，建议过表达全长，并截短和 草地贪夜蛾中这些半胱氨酸蛋白酶的突变衍生物 (Sf9)细胞和E.杆菌这将有助于大量的这些 用于功能和结构研究的蛋白质。哺乳动物细胞凋亡 在我们的实验室克隆的半胱氨酸蛋白酶，将进行表征。他们的 将分析基因的组织特异性和时间表达， 选择性剪接异构体的存在。潜在底物和 这些蛋白酶的抑制剂将使用生物化学和 分子生物学技术预计这些研究将 有助于阐明这一重要的激活机制， 蛋白酶类。这将加强努力， 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中的分子机制， 内稳态，拟议的研究也将产生重要的知识 可以应用于治疗许多人类疾病， 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.