Jones M E, Haire M F, Kloetzel P M, Mykles D L, Schwartz L M
Department of Biology, University of Massachusetts, Amherst 01003, USA.
Dev Biol. 1995 Jun;169(2):436-47. doi: 10.1006/dbio.1995.1159.
The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta are a well-characterized model system for examining the biochemical changes that accompany programmed cell death during development. These giant muscles die during a 30-hr period in response to a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. When the ISMs become committed to die, there are dramatic increases in both ubiquitin expression and ubiquitin-dependent proteolysis. Since the multicatalytic proteinase (MCP) is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its composition and properties. The purified enzyme from whole larval integumentary tissues resembles MCPs isolated from other species with respect to subunit composition and general catalytic properties. However, when MCP was isolated from condemned ISMs, we observed an approximately ninefold increase in proteinase activity compared to MCP from precommitment muscles. This increase in proteolytic activity was correlated with an approximately eightfold increase in the absolute amounts of MCP protein as determined by Western blotting and densitometry. When purified MCP from condemned muscles was examined by two-dimensional polyacrylamide gel electrophoresis, four new subunits that were not detected in the precommitment muscles were present. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. These data are consistent with the hypothesis that when the ISMs become committed to die, more MCP accumulates in cells and new subunits are synthesized that change both the enzymatic properties and the conformation of MCP, which in turn participates in the dramatic proteolysis that accompanies cell death.
烟草天蛾Manduca sexta的节间肌（ISMs）是一个特征明确的模型系统，用于研究发育过程中伴随程序性细胞死亡的生化变化。这些巨大的肌肉在30小时内死亡，以应对昆虫蜕皮激素20-羟基蜕皮酮循环滴度的下降。当ISMs开始走向死亡时，泛素表达和泛素依赖性蛋白水解都显著增加。由于多催化蛋白酶（MCP）负责细胞中ATP/泛素依赖性蛋白水解，我们研究了其组成和特性。从整个幼虫体表组织中纯化的酶在亚基组成和一般催化特性方面与从其他物种中分离的MCP相似。然而，当从注定死亡的ISMs中分离MCP时，我们观察到与未注定死亡的肌肉中的MCP相比，蛋白酶活性增加了约九倍。这种蛋白水解活性的增加与通过蛋白质印迹法和光密度测定法确定的MCP蛋白绝对量增加约八倍相关。当通过二维聚丙烯酰胺凝胶电泳检测从注定死亡的肌肉中纯化的MCP时，发现了在未注定死亡的肌肉中未检测到的四个新亚基。与这些新亚基的添加相关的是，在垂死肌肉的整个细胞质和细胞核内，免疫可检测的MCP水平显著增加。MCP的这些变化受介导细胞死亡的相同激素信号调节。这些数据与以下假设一致：当ISMs开始走向死亡时，更多的MCP在细胞中积累，并且合成了新的亚基，这些亚基改变了MCP的酶学性质和构象，进而参与了伴随细胞死亡的剧烈蛋白水解过程。
