Analyses of molecular mechanisms of caspases during neural development

凋亡相关基因如caspase-3、caspase-9和Apaf-1作为细胞死亡诱导剂具有重要作用。然而，一些证据表明，不诱导细胞死亡的caspase-3和caspase-9基因敲除小鼠的神经发育和分化需要caspase-3和caspase-9的轻微激活。结果1.神经发育过程中caspase-7表达的鉴定：在原代培养的早期，caspase-7表达增加，而caspase-3在神经分化过程中表达增加。将人caspase-3和caspase-7基因分别克隆到pET表达载体中，在大肠杆菌中诱导表达。大肠杆菌，3.半胱氨酸蛋白酶底物的筛选7.小池cDNA文库每个池含有约100个人脑基因，共17，000个基因。在活性半胱天冬酶存在下，使用355标记的蛋白质通过体外切割测定筛选底物，所述蛋白质通过体外转录和翻译产生。放射自显影显示caspase-7的裂解产物。4.人脑cDNA中caspase-7底物的鉴定从96个库cDNA文库中鉴定出18个基因为caspase-7底物。18个基因中有5个是未知基因，10个基因包含全长，2个质粒没有编码区。我们将分析神经发育基质的功能。

Apoptotic related genes, such as caspase-3, caspase-9, and Apaf-1, have a critical roles as cell death inducer. However, several evidences suggest that trivial activation of caspases, which do not induce cell death, needs to neural development and differentiation because knockout mice of caspase-3 and caspase-9 show disorganized neural development. In this project, we planned to identify caspases to be activated during neural development and screen its substrates to analyze the molecular roles of caspases.Results1.Identification of caspases expression during neural development.The expression of caspase-7 was increased at early stage of primary neuronal culture by RT-PCR, whereas caspase-3 was increased during neural differentiation.2.Generation of active caspase in E. coli.Human caspase-3 and caspase-7 genes were cloned into pET expression vectors and active caspases were induced in E. coli., and purified.3.Screening of substrates for caspase-7.Small pool cDNA library contained about 100 genes from human brain per pool, which comes to 17,000 genes in total. Substrates were screened by in vitro cleavage assay in the presence of active caspase using 355-labeled proteins, which were generated by in vitro transcription and translation. Autoradiograph showed the cleaved product by caspase.4.Identification of caspase-7 substrates from human brain cDNA.Eighteen genes were identified as caspase-7 substrates from 96 pools cDNA library. Five of eighteen were unknown genes, ten genes included full-length, and two plasmids had no coding region. We are going to analyze the function of substrates for neural development.