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

caspase蛋白酶家族在脊椎动物细胞凋亡的实施中起着核心作用。半胱天冬酶在健康细胞中组成性表达，在那里它们被合成为前体蛋白（原半胱天冬酶）。半胱天冬酶在原半胱天冬酶的加工过程中被激活。因此，控制caspase激活的调控机制对细胞凋亡的调控具有重要意义。细胞生物学研究已经表明，caspase-12的活化是由内质网（ER）损伤特异性诱导的，但caspase-12活化的调控机制尚不清楚。内质网应激受到越来越多的关注，因为它被认为是病理相关的细胞凋亡的原因。我们通过酵母双杂交筛选从HeLa细胞cDNA文库中分离出人类癌症抗原MAGE-3，作为特异性结合procaspase-12的蛋白。MAGE-3是MAGE（黑色素瘤相关抗原）基因家族的一员，在多种肿瘤中表达，但在除睾丸外的正常组织中不表达。与MAGE-3与procaspase-12的结合一致，MAGE-3通过特异性结合前体来保护procaspase-12免受加工。为了研究MAGE-3对细胞中caspase-12激活的影响，我们建立了稳定的过表达MAGE-3的转染物。Western blot分析显示，MAGE-3的稳定表达使procaspase-12对内质网应激不敏感，从而抑制细胞凋亡。尽管亲本细胞在内质酰胺胁迫诱导剂的作用下发生凋亡，但稳定的转染物对诱导剂具有抗性，在未处理的细胞中观察到相同的低背景水平发生凋亡。尽管目前尚不清楚MAGE-3是否在肿瘤细胞的caspase调节中发挥任何作用，但我们的初步数据显示，到目前为止，在几种肿瘤细胞系（例如Jurkat）的微粒体部分检测到内源性MAGE-3。我们的研究结果还表明，反义MAGE-3的过表达使Jurkat细胞对内质网应激的抗性降低，而义结构对其抗性没有影响。这些结果支持了MAGE-3在肿瘤细胞中的特异性表达可能参与肿瘤细胞对内质网应激的抵抗。研究MAGE-3和procaspase-12之间的特异性相互作用可能为开发治疗内质网应激引起的caspase活化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