Subcellular localization of the proteins implicated in DNA damage-induced cell death

细胞凋亡或程序性细胞死亡是一个受进化保守基因调控的生理过程。细胞凋亡的失调可导致许多重大疾病，包括癌症、自身免疫性疾病和某些神经退行性疾病。DNA损伤诱导的细胞凋亡是由Bcl-2家族的促凋亡成员（如Bim）和半胱氨酸蛋白酶家族（称为caspases）完成的。在两年的时间里，我们得到了如下的结果。我们克隆并鉴定了6种新的人类Bim同工异构体，分别为Bimα1、α2和β1-β4，它们是通过选择性剪接产生的。在这些新型异构体中，只有bm α1和α2含有BH3结构域，具有促进细胞凋亡的作用，但其作用弱于经典异构体。这两种同工异构体至少部分定位于线粒体。至少在人类中，bim亚型的表达谱在正常组织中是高度可变的，这表明bim具有组织特异性的转录调控。Caspase-8和-10以蛋白酶不依赖的方式诱导NF-κB活化。利用GST下拉实验研究导致caspase-8和-10介导的NF-κB激活的信号通路发现，在激活NF-κB的上游激酶中，NIK和RIP可以直接结合caspase-8和-10，而RICK和IKKα/β不能。通过使用显性阴性突变体和小干扰RNA技术，我们也证明了NIK和IKKα在这一过程中是必需的。

Apoptosis or programmed cell death is a physiologic process that is regulated by evolutionarily conserved genes. Dysregulation of apoptosis can contribute to many major diseases including cancer, autoimmune disorders and certain neurodegenerative diseases. DNA damage-induced apoptosis is accomplished by proapoptotic members of the Bcl-2 family, such as Bim, and a family of cysteine proteases termed caspases. During the period of two years we got the results as follows.We have cloned and characterized six novel isoforms of human Bim, designated as Bimα1, α2, and β1-β4, which are generated by alternative splicing. Among the novel isoforms, only Bimα1 and α2 contained a BH3 domain and were proapoptotic, although less potent than the classical isoforms. These two isoforms localized, at least in part, in mitochondria. Expression profiles of bim isoforms were highly variable among normal tissues at least in humans, suggesting a tissue-specific transcriptional regulation of bim.Caspase-8 and -10 can induce NF-κB activation in protease-independent manner. Investigation of the signaling pathways leading to caspase-8 and -10-mediated NF-κB activation using the GST pull-down assay revealed that, among upstream kinases that activate NF-κB, NIK and RIP, but not RICK or IKKα/β could directly bind to caspase-8and -10. By using dominant-negative mutants and small interfering RNA technology, we also demonstrated that NIK and IKKα are required for this process.