Lazebnik Y A, Takahashi A, Moir R D, Goldman R D, Poirier G G, Kaufmann S H, Earnshaw W C
Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9042-6. doi: 10.1073/pnas.92.20.9042.
Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1 beta-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enyzme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus.
尽管特定蛋白酶在细胞凋亡的活跃阶段起着关键作用，但其底物大多未知。我们之前鉴定出聚（ADP - 核糖）聚合酶（PARP）是与白细胞介素1β转换酶（ICE）相关的蛋白酶的凋亡相关底物。现在我们使用无细胞系统来表征在细胞凋亡期间切割核纤层蛋白的蛋白酶。细胞凋亡期间的核纤层蛋白切割需要第二种ICE样酶的作用，该酶表现出切割动力学以及对特定抑制剂的敏感性特征，这与PARP蛋白酶不同。因此，这些无细胞提取物中的凋亡事件需要多种ICE样酶。用甲苯磺酰赖氨酸“氯甲基酮”抑制核纤层蛋白酶可在浓缩染色质包装成凋亡小体之前阻断核凋亡。在这些条件下，核DNA被完全切割成核小体梯状条带。我们的研究表明，在凋亡执行的最后阶段，核纤层蛋白酶和片段化核酸酶在独立的平行途径中发挥作用。单独任何一条途径都不足以完成核凋亡。相反，各种活性协同作用以驱动细胞核的解体。
