乙酰胆碱酯酶在细胞凋亡中的作用及G418诱导细胞凋亡的分子机制

【作者】 金其煌；

【导师】 张学军；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 分子细胞生物学， 2004， 博士

【摘要】 哺乳动物的多种细胞在多种条件诱导凋亡过程中会表达乙酰胆碱酯酶(acetylcholinesterase,AChE),用反义核酸抑制 AChE 的表达可以抑制一部分细胞的凋亡,但 AChE 在凋亡中的功能仍不清楚。为研究其可能的功能,我们通过转基因方法在 NRK(Normal Rat Kidney)细胞中过表达 AChE。正常 NRK 细胞中检测不到 AChE 的酯酶活性,但用 Western blot 方法可检测到 AChE 蛋白的存在。在多种细胞凋亡诱导因子刺激下,凋亡的 NRK 细胞中出现了 AChE 酯酶活性,并且 AChE 的 mRNA 和蛋白也随着增加。在 NRK 细胞中过表达 AChE 蛋白没有诱导细胞凋亡,并且我们得到了稳定表达的细胞株,但这种细胞株的生长明显延缓了,而用稳定表达 AChE 反义核酸抑制 AChE 蛋白的表达却能促进细胞的生长。在无血清诱导下,MTT 实验结果显示 AChE 表达株的残留细胞活性比对照的低,而 AChE 反义核酸表达株的残留细胞活性比对照的高。这些结果说明在凋亡过程中 AChE 有两个作用,先是增加的 AChE 抑制细胞的生长,然后是促进细胞凋亡的进程。为检测 NRK 细胞中的无酯酶活性 AChE 在凋亡中可能的功能,我们用一种蛋白合成的抑制剂 G418 来诱导细胞凋亡。G418 诱导的凋亡 NRK 细胞中有非常明显的 AChE 酯酶活性,而这种 AChE 酯酶活性不依赖于新蛋白的合成和 caspases 的活性。用共聚焦显微镜观察 AChE 的细胞定位,观察到正常 NRK细胞中 AChE 定位于内质网上,而在凋亡的细胞中 AChE 分布于整个细胞。用透射电镜观察 AChE 酯酶活性的产物分布,发现产物主要定位在细胞核中,并有一<WP=5>部分位于胞质和一些囊泡的外膜上。缺失内质网定位信号肽的 AChE 表达质粒使在胞质直接表达的 AChE 不能诱导细胞凋亡。但长时间培养后,在胞质中表达AChE 细胞的凋亡比例明显比在内质网中表达 AChE 细胞的高。有趣的是在胞质中表达 AChE 蛋白的 C 末端肽片段也有促进细胞凋亡的功能。根据上述结果我们提出一个假设:在 NRK 细胞中无酯酶活性的 AChE 在内质网中比较稳定地存在,当受到凋亡诱导因子刺激后以某种机制从内质网转移到胞质并部分转到细胞核内,在胞质和核中其 C 末端肽段同其它的凋亡因子相互作用并促进细胞凋亡,同时 AChE 本身的构象也发生改变,成为有酯酶活性的形式。为进一步分析无酯酶活性 AChE 蛋白,我们制备了 AChE 抗体亲和层析柱,分离出总的 AChE 蛋白。我们还制备了以 AChE 抑制剂(tacrine)为配体的亲和层析柱,分离有酯酶活性的AChE。这两个方法为在蛋白水平上比较无酯酶活性和有酯酶活性 AChE 之间的差异奠定了基础。更多还原

【Abstract】 Acetylcholinesterase (AChE) is expressed in a number of cell lines uponinduction of apoptosis by various stimuli, and inhibiting expression of AChE byAChE antisense prevents part of the cells from apoptosis. However, the function ofAChE in apoptosis is still elusive. We made AChE overexpresse in Normal RatKidney (NRK) cells to investigate its possible function. AChE activity was notpresented in living NRK cells, but the AChE protein existed. Upon induction ofapoptosis, apoptotic NRK cells presented AChE activity, and AChE mRNA andprotein also increased. Overexpression of AChE in NRK cells did not induceapoptosis. However, the proliferation of cell lines expressing AChE was retarded, andthe effect was reversed in cell lines overexpressing AChE antisene, in which AChEexpression decreased. Under induction by serum deprivation, the residual cell activityof cells overexpressing AChE was lower than control, and the effect was reversed incell lines expressing AChE antisense. It suggested that AChE had two functions in theprocess of apoptosis: increased AChE protein first inhibited cell proliferation, andthen promoted apoptosis. To detect the possible function of inactive AChE duringapoptosis, NRK cells were induced apoptosis by G418, which is an inhibitor ofprotein synthesis. Apoptotic NRK cells induced by G418 exhibited strong AChEactivity, which was independent of new protein synthesis and capases activity. Inliving NRK cells, AChE protein located in ER, and in apoptotic cells, AChEdistributed over the whole cell. The products of AChE enzymatic reaction observedunder electron microscope were located in nuclei, cytosole, and on the membrane ofsome vacuoles. Expression of AChE without ER location signal (ERLS) did not<WP=7>induce apoptosis. However, the percentage of apoptotic cells in NRK cells transfectedwith AChE without ERLS was higher than that in NRK cells transfected with intactAChE. Interestingly, cells expressed C-terminal of AChE had the same effect withcells expressed AChE without ERLS, and moreover, AChE directly expressed incytosole had no activity. Base on these experiments, we brought forward a hypothesison AChE in apoptosis: in the process of apoptosis, inactive AChE, which stably stoodin ER in NRK cells, was translocated into cytosole and nuclei, where the C-terminalof AChE interacted with some apoptotic factors and promoted apoptosis, and at thesame time, inactive AChE was transformed into active AChE. To further analyse theinactive AChE protein, we prepared antibody affinity chromatography to isolate thetotal AChE. This method with tacrine affinity chromatography which was to isolateactive AChE provided precondition for analysis of the difference between inactive andactive AChE at the protein level.更多还原