Identification of the endoplasmic reticulum stress-specific caspase and its regulators in human

半胱天冬酶蛋白酶家族在脊椎动物细胞凋亡的实施中起着核心作用。胱天蛋白酶在健康细胞中组成型表达，在那里它们被合成为前体蛋白（胱天蛋白酶原）。半胱天冬酶在加工半胱天冬酶原时被激活。细胞生物学研究表明，caspase-12的激活是由内质网（endoplasmic reticulum，ER）损伤引起的，但caspase-12激活的调控机制尚不清楚。内质网应激被认为是病理性细胞凋亡的一个重要原因，因此受到越来越多的关注。我们已经通过酵母双杂交筛选从HeLa细胞cDNA文库中分离了人癌抗原法师-3，作为特异性结合半胱氨酸蛋白酶原-12的蛋白。法师-3是法师（melanoma associated antigen，黑色素瘤相关抗原）基因家族的成员，在多种肿瘤细胞中表达。 ...更多信息 在睾丸以外的正常组织中不存在。与法师-3与半胱氨酸天冬氨酸蛋白酶原-12的结合一致，法师-3通过特异性结合前体来保护半胱氨酸天冬氨酸蛋白酶原-12免受加工。为了检测法师-3对细胞中半胱天冬酶-12活化的作用，我们建立了过表达法师-3的稳定转染子。Western blot分析表明，稳定表达的法师-3提供procaspase-12不敏感的ER应力，从而抑制凋亡。尽管亲本细胞响应于ER应激诱导剂而经历凋亡，但稳定的转染子对诱导剂具有抗性，在未处理细胞中观察到的相同低背景水平下经历凋亡。尽管仍不清楚法师-3是否在肿瘤细胞中的半胱天冬酶调节中起任何作用，但我们的初步数据显示，在几种肿瘤细胞系（例如，朱熹，至今已考。我们的研究结果还表明，过表达反义法师-3使Jurkat细胞对ER应激的抵抗力降低，而正义构建体并不影响抵抗力。这些结果支持肿瘤细胞特异性表达法师-3可能参与肿瘤细胞抵抗ER应激的理论。研究法师-3和半胱天冬酶原-12之间的特异性相互作用可能为开发针对由ER应激引起的不必要的半胱天冬酶激活的治疗试剂提供基础。少

The caspase protease family plays a central role in 'the implementation of apoptosis in vertebrates. Caspases are constitutively expressed in healthy cells, where they are synthesized as precursor proteins (procaspases). Caspases are activated upon processing of procaspases. Therefore, the regulatory mechanism that controls caspase activation is significant for regulation of apoptosis.Cell biological studies have already revealed that activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER), yet the regulatory mechanism of caspase-12 activation have been unclear. ER stress has received growing attention because it is considered a cause of pathologically relevant apoptosis. We have isolated by yeast two-hybrid screening from a HeLa cell cDNA library a human cancer antigen, MAGE-3,as a protein that specifically binds the procaspase-12. MAGE-3 is a member of the MAGE (melanoma associated antigen) gene family and is expressed in variou … More s types of tumor but not in normal tissues except for the testis. Consistent with the binding of MAGE-3 to procaspase-12, MAGE-3 protects procaspase-12 from processing by specifically binding the precursor. To examine the effect of MAGE-3 on caspase-12 activation in cells, we established stable transfectants that overexpress MAGE-3. Western blot analyses showed that stable expression of MAGE-3 rendered procaspase-12 insensitive to ER stress, thereby suppressing apoptosis. Although the parental cells underwent apoptosis in response to ER stress inducers, the stable transfectants were resistant to the inducers, undergoing apoptosis at the same low background level observed in untreated cells.Although it remains unclear whether MAGE-3 plays any role in caspase regulation in tumor cells, our preliminary data show that endogenous MAGE-3 is detected in the microsomal fraction in several tumor cell lines (e.g., Jurkat) so far examined. Our results also show that overexpression of antisense MAGE-3 rendered Jurkat cells less resistant to ER stress, whereas the sense construct did not affect the resistance. These results support the theory that specific expression of MAGE-3 in tumor cells may be involved in resistance of tumor cells to ER stress. A study of the specific interactions between MAGE-3 and procaspase-12 may provide a basis for the development of therapeutic reagents against unwanted activation of caspases caused by ER stress. Less