Cain K, Bratton S B, Langlais C, Walker G, Brown D G, Sun X M, Cohen G M
MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester, LE1 9HN United Kingdom.
J Biol Chem. 2000 Mar 3;275(9):6067-70. doi: 10.1074/jbc.275.9.6067.
Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large in the order of approximately 1.4 MDa. We now demonstrate that dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M(r) values of approximately 1.4 MDa and approximately 700 kDa. Kinetic analysis demonstrates that in vitro the approximately 700-kDa complex is produced more rapidly than the approximately 1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the approximately 700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processed effector caspases. Thus, the approximately 700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis.
凋亡蛋白酶激活因子-1(Apaf-1)通过与半胱天冬酶-9结合并激活它,在细胞凋亡过程中发挥关键作用。在dATP和细胞色素c存在的情况下,Apaf-1的寡聚化是激活半胱天冬酶-9所必需的,并产生一个激活半胱天冬酶的凋亡小体复合物。重组蛋白的重组研究表明,该复合物的大小非常大,约为1.4兆道尔顿。我们现在证明,细胞裂解物的dATP激活导致形成两种含有Apaf-1的大型凋亡小体复合物,其相对分子质量分别约为1.4兆道尔顿和700千道尔顿。动力学分析表明,在体外,约700千道尔顿的复合物比约1.4兆道尔顿的复合物产生得更快,并且表现出更强的激活效应半胱天冬酶的能力。值得注意的是,在用依托泊苷或N-甲苯磺酰-L-苯丙氨酰氯甲基酮(TPCK)处理后经历凋亡的人肿瘤单核细胞中,主要形成了约700千道尔顿的含有Apaf-1的凋亡小体复合物。该复合物加工效应半胱天冬酶。因此,约700千道尔顿的复合物似乎是在细胞凋亡过程中组装形成的正确且具有生物活性的凋亡小体复合物。