A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K+ deprivation.
A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K+ deprivation.
复制标题
除 CPP32 外，DEVD 抑制的 caspase 参与小脑颗粒神经元 K 剥夺诱导的细胞凋亡。
DOI：
10.1046/j.1471-4159.1998.70051809.x
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发表时间：
1998
影响因子：
4.7
通讯作者：
Haycock,JW
中科院分区：
文献类型：
--
作者：
D'Mello,SR;Aglieco,F;Roberts,MR;Borodezt,K;Haycock,JW
培养的小脑颗粒神经元在从含有去极化K+的培养液(25mMKCI)切换到含有较低K+的培养液(5mMKCI)时发生凋亡。我们使用这个范式来研究半胱氨酸酶在死亡过程中的作用。两种广谱半胱氨酸天冬氨酸氨基转移酶抑制剂tert‐butoxycarbonyl‐Asp·(O‐methyl)·fluoromethyl酮和苯氧羰基-丙氨酸-天冬氨酸·氟甲基酮在较低浓度(10-25µM)下显著降低细胞死亡率(分别为90%和60%)，提示半胱氨酸天冬氨酸酶的激活参与了细胞的凋亡过程。这些caspase抑制剂也减少了DNA片段化，这是细胞凋亡的标志，表明caspase激活发生在导致细胞死亡的一系列事件中DNA裂解的上游。作为确定参与caspase(S)的一步，还评价了白介素1β转换酶选择性抑制剂N-乙酰酪氨酸-丙氨酸-天冬氨酸·氯甲基酮(YVAD.CMK)和CPP32选择性抑制剂N-乙酰天冬氨酸-谷氨酸-天冬氨酸·氟甲基酮(DEVD.FMK)的作用。YVAD·CMK仅提供温和(20%)的保护，且仅在测试的最高浓度(100uM)下提供保护，表明IL-1β转换酶和/或密切相关的caspase不参与其中。相比之下，DEVD·fmk对细胞死亡的抑制率高达50%。然而，Western印迹分析未能检测到在诱导颗粒神经元凋亡过程中CPP32的加工/激活或CPP32底物聚(ADP-核糖)聚合酶的蛋白分解增加。同样，Nedd2的水平也没有受到影响。Nedd2是一种在大脑中高表达的半胱氨酸天冬氨酸氨基转移酶，它被DEVD·fmk部分抑制。这些结果表明，除CPP32或Nedd2外，对DEVD敏感的caspase介导了K+剥夺颗粒神经元的凋亡诱导。
Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+(25 mMKCI) to medium containing lower levels of K+(5 mMKCI). We used this paradigm to investigate the role of caspases in the death process. Two broad‐spectrum caspase inhibitors,tert‐butoxycarbonyl‐Asp·(O‐methyl)·fluoromethyl ketone and benzyloxycarbonyl‐Val‐Ala‐Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects ofN‐acetyl Tyr‐Val‐Ala‐Asp·chloromethyl ketone (YVAD·cmk), an interleukin‐1β converting enzyme‐preferring inhibitor, andN‐acetyl Asp‐Glu‐Val‐Asp·fluoromethyl ketone (DEVD·fmk), a CPP32‐preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin‐1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP‐ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD‐sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+‐deprived granule neurons.