猕猴脑胱天蛋白酶-3活化及其靶蛋白的体外研究 |
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引用本文: | 张爱群,初向阳,赖惠玲,武艳,姚大卫. 猕猴脑胱天蛋白酶-3活化及其靶蛋白的体外研究[J]. 生物化学与生物物理进展, 2002, 29(6) |
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作者姓名: | 张爱群 初向阳 赖惠玲 武艳 姚大卫 |
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作者单位: | 1. 解放军总医院老年医学研究所,北京,100853;香港中文大学解剖系,新界沙田,香港 2. 解放军总医院胸外科,北京,100853 3. 香港中文大学解剖系,新界沙田,香港 |
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摘 要: | 凋亡的主要生化过程包括胱天蛋白酶的活化及其对细胞内蛋白质的选择性切割.在已知的胱天蛋白酶中,可被多种凋亡刺激信号激活的胱天蛋白酶-3备受注目.为进一步揭示灵长类动物神经组织中未知的胱天蛋白酶-3靶蛋白,采用成年猕猴脑组织粗提物作为无细胞体系,通过加入granzyme B引发凋亡途径的部分反应,如胱天蛋白酶-3的活化及随后发生的蛋白质水解.经蛋白质印迹分析发现,与granzyme B共孵育后,猕猴脑胱天蛋白酶-3以两步方式从酶原转化为活性酶.对猕猴脑组织自身蛋白质的进一步分析显示,多聚ADP-核糖聚合酶(PARP)被水解为长85 ku的片段,此片段提示胱天蛋白酶-3的特异切割活性.此外,神经元凋亡抑制蛋白(NAIP)也被切割,产生长约40 ku的小片段,但是它的出现不被胱天蛋白酶-3特异性抑制剂Ac-DEVD-CHO阻断,因此可能是granzyme B直接作用于NAIP所致.以上结果提示,凋亡相关酶切反应可在成年猕猴脑组织提取物中得到重现;NAIP可能是granzyme B而非胱天蛋白酶-3的作用靶点.
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关 键 词: | 凋亡 granzyme B 胱天蛋白酶-3 多聚ADP-核糖聚合酶 神经元凋亡抑制蛋白 灵长类 脑 |
In vitro Investigation of Caspase-3 Activation and Its Proteolytic Targets in Adult Monkey Brain |
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Abstract: | The major biochemieal process of apoptosis involves the activation of a group of proteases (caspases) and the selective cleavage of a set of intracellular proteins that leads to the collapse of cell survival mechanism. Among the caspases identified, caspase-3 stands out because it is conmonly activated by numerous death signals and cleaves a growing number of cellular conponents. In order to reveal potential targets of easpase-3 in primate neural tissue, an alternative cell free system based on adult monkey brain was established to reproduce the downstream part of apoptotic program, initiated by the addition of granzyme B. Through Western blot analysis, caspase-3 was found to become mature in a two-step manner and its activity was exhibited by the cleavage of the synthetic substrate, Ac-DEVD-pNA. Investigations on native proteins in the brain extract showed that poly (ADP-ribose) polymerase (PARP) was cleaved to an 85 ku fragment, suggestive of caspase-3 activity. And more intriguingly, a neuronal apoptosis inhibitory protein (NAIP)-irnmunoreactive fragment with molecular mass of approximately 40 ku was detected in granzyme B-treated brain extract and its production was not blocked by the caspase-3 inhibitor, Ac-DEVD-CHO. According to the substrate specificity of granzyme B and the size of cleavage product, putative cleavage site may be located immediately after the third DIR domain of NAIP. These data suggest that cleavage events involved in apoptosis can be reproduced in matured primate brain extract and NAIP is likely to be the target of granzyme B, but not of caspase-3, during apoptosis. |
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Keywords: | atoptosis granzyme B caspase-3 poly(ADP-ribose)poly merase apoptosis inhibitory protein primate brain |
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