Armstrong R C, Aja T J, Hoang K D, Gaur S, Bai X, Alnemri E S, Litwack G, Karanewsky D S, Fritz L C, Tomaselli K J
IDUN Pharmaceuticals, Inc., La Jolla, California 92037, USA.
J Neurosci. 1997 Jan 15;17(2):553-62. doi: 10.1523/JNEUROSCI.17-02-00553.1997.
Neuronal apoptosis occurs during nervous system development and after pathological insults to the adult nervous system. Inhibition of CED3/ICE-related proteases has been shown to inhibit neuronal apoptosis in vitro and in vivo, indicating a role for these cysteine proteases in neuronal apoptosis. We have studied the activation of the CED3/ICE-related protease CPP32 in two in vitro models of mouse cerebellar granule neuronal cell death: K+/serum deprivation-induced apoptosis and glutamate-induced necrosis. Pretreatment of granule neurons with a selective, irreversible inhibitor of CED3/ICE family proteases, ZVAD-fluoromethylketone, specifically inhibited granule neuron apoptosis but not necrosis, indicating a selective role for CED3/ICE proteases in granule neuron apoptosis. Extracts prepared from apoptotic, but not necrotic, granule neurons contained a protease activity that cleaved the CPP32 substrate Ac-DEVD-aminomethylcoumarin. Induction of the protease activity was prevented by inhibitors of RNA or protein synthesis or by the CED3/ICE protease inhibitor. Affinity labeling of the protease activity with an irreversible CED3/ICE protease inhibitor, ZVK(biotin)D-fluoromethylketone, identified two putative protease subunits, p20 and p18, that were present in apoptotic but not necrotic granule neuron extracts. Western blotting with antibodies to the C terminus of the large subunit of mouse CPP32 (anti-CPP32) identified p20 and p18 as processed subunits of the CPP32 proenzyme. Anti-CPP32 specifically inhibited the DEVD-amc cleaving activity, verifying the presence of active CPP32 protease in the apoptotic granule neuron extracts. Western blotting demonstrated that the CPP32 proenzyme was expressed in granule neurons before induction of apoptosis. These results demonstrate that the CED3/ICE homolog CPP32 is processed and activated during cerebellar granule neuron apoptosis. CPP32 activation requires macromolecular synthesis and CED3/ICE protease activity. The lack of CPP32 activation during granule neuron necrosis suggests that proteolytic processing and activation of CED3/ICE proteases are specific biochemical markers of apoptosis.
神经元凋亡发生在神经系统发育过程中以及成体神经系统受到病理损伤之后。研究表明，抑制CED3/ICE相关蛋白酶在体外和体内均能抑制神经元凋亡，这表明这些半胱氨酸蛋白酶在神经元凋亡中发挥作用。我们在小鼠小脑颗粒神经元死亡的两种体外模型中研究了CED3/ICE相关蛋白酶CPP32的激活情况：钾离子/血清剥夺诱导的凋亡和谷氨酸诱导的坏死。用CED3/ICE家族蛋白酶的选择性、不可逆抑制剂ZVAD-氟甲基酮预处理颗粒神经元，可特异性抑制颗粒神经元凋亡，但不抑制坏死，这表明CED3/ICE蛋白酶在颗粒神经元凋亡中具有选择性作用。从凋亡而非坏死的颗粒神经元制备的提取物含有一种蛋白酶活性，该活性可切割CPP32底物Ac-DEVD-氨基甲基香豆素。RNA或蛋白质合成抑制剂或CED3/ICE蛋白酶抑制剂可阻止蛋白酶活性的诱导。用不可逆的CED3/ICE蛋白酶抑制剂ZVK(生物素)D-氟甲基酮对蛋白酶活性进行亲和标记，鉴定出两个推定的蛋白酶亚基p20和p18，它们存在于凋亡而非坏死的颗粒神经元提取物中。用针对小鼠CPP32大亚基C末端的抗体（抗CPP32）进行蛋白质印迹分析，确定p20和p18为CPP32酶原的加工亚基。抗CPP32特异性抑制DEVD-amc切割活性，证实凋亡颗粒神经元提取物中存在活性CPP32蛋白酶。蛋白质印迹分析表明，CPP32酶原在凋亡诱导前在颗粒神经元中表达。这些结果表明，CED3/ICE同源物CPP32在小脑颗粒神经元凋亡过程中被加工和激活。CPP32的激活需要大分子合成和CED3/ICE蛋白酶活性。颗粒神经元坏死过程中缺乏CPP32激活表明，CED3/ICE蛋白酶的蛋白水解加工和激活是凋亡的特异性生化标志物。
