Chen D, Stetler R A, Cao G, Pei W, O'Horo C, Yin X M, Chen J
Departments of Neurology, Pittsburgh Institute for Neurodegenerative Disorders, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
J Biol Chem. 2000 Dec 8;275(49):38508-17. doi: 10.1074/jbc.M003906200.
Nuclear changes, including internucleosomal DNA fragmentation, are classical manifestations of apoptosis for which the biochemical mechanisms have not been fully elucidated, particularly in neuronal cells. We have cloned the rat DNA fragmentation factor 35/inhibitor of caspase-activated DNase (short form) (DFF35/ICAD(S)) and found it to be the predominant form of ICAD present in rodent brain cells as well as in many other types of cells. DFF35/ICAD(S) forms a functional complex with DFF40/caspase-activated DNase (CAD) in the nucleus, and when its caspase-resistant mutant is over-expressed, it inhibits the nuclease activity, internucleosomal DNA fragmentation, and nuclear fragmentation but not the shrinkage and condensation of the nucleus, in neuron-differentiated PC12 cells in response to apoptosis inducers. DFF40/CAD is found to be localized mainly in the nucleus, and during neuronal apoptosis, there is no evidence of further nuclear translocation of this molecule. It is further suggested that inactivation of DFF40/CAD-bound DFF35 and subsequent activation of DFF40/CAD during apoptosis of neuronal cells may not occur in the cytosol but rather in the nucleus through a novel mechanism that requires nuclear translocation of caspases. These results establish that DFF35/ICAD(S) is the endogenous inhibitor of DFF40/CAD and caspase-dependent apoptotic DNA fragmentation in neurons.
核变化，包括核小体间DNA片段化，是细胞凋亡的经典表现，其生化机制尚未完全阐明，尤其是在神经元细胞中。我们克隆了大鼠DNA片段化因子35/半胱天冬酶激活的脱氧核糖核酸酶抑制剂（短形式）（DFF35/ICAD(S)），发现它是啮齿动物脑细胞以及许多其他类型细胞中ICAD的主要形式。DFF35/ICAD(S)在细胞核中与DFF40/半胱天冬酶激活的脱氧核糖核酸酶（CAD）形成功能复合物，当其抗半胱天冬酶突变体过表达时，它会抑制核酸酶活性、核小体间DNA片段化和核片段化，但不会抑制神经元分化的PC12细胞中细胞核在凋亡诱导剂作用下的收缩和凝聚。发现DFF40/CAD主要定位于细胞核，并且在神经元凋亡过程中，没有证据表明该分子会进一步发生核转位。进一步表明，在神经元细胞凋亡过程中，与DFF40/CAD结合的DFF35的失活以及随后DFF40/CAD的激活可能不是在细胞质中发生，而是在细胞核中通过一种需要半胱天冬酶核转位的新机制发生。这些结果表明DFF35/ICAD(S)是神经元中DFF40/CAD和半胱天冬酶依赖性凋亡DNA片段化的内源性抑制剂。
