BASIC STUDIES ON A TRANSCRIPTION FACTOR NF-κB AND SIGNAL TRANSDUCTION THERAPY

核因子-κB控制着许多基因的表达，最近发现它参与了抑制细胞凋亡。核因子-κB通过与其他细胞蛋白相互作用发挥其生物学作用。为了确定分子伴侣，我们应用了酵母双杂交筛选，以部分NF-κB p65亚基(RELA)为“诱饵”。我们已经分离到一个新的基因，称为RELA相关抑制物(RAI)，随后证明它位于细胞核内，并通过抑制其DNA结合来阻断NF-κB介导的基因表达。因此，RAI似乎作为一种故障安全机制来确保NF-κB的沉默，或者作为一种反馈系统来下调NF-κB的活性。使用p65的不同部分作为“诱饵”，我们已经鉴定出AES和TLE1，这是Groucho家族蛋白的成员，被认为是果蝇人类Rela，Backal同源物的转录共抑制因子。我们已经证明了人的aES/TLE1也阻断了NF-κB介导的转录，因为最近发现NF-κB参与了高等多细胞生物的形态发生，预计人的Groucho同源物可能与NF-κB一起参与人体计划，尽管确切的机制和动力学还有待进一步探索。最后，我们已经确定53BP2，最初被确定为肿瘤抑制蛋白P53的相互作用分子伙伴，作为与P65相互作用的蛋白。当53BP2被转导到细胞内时，这些细胞被杀伤，并具有提示细胞凋亡的特征，如核碎裂和浓缩，以及Annexin V染色。有趣的是，当p65基因与53BP2共转染时，53BP2诱导的细胞死亡完全被阻断。这些发现表明，P65与53BP2的相互作用可能至少在一定程度上解释了NF-κB和P65(RERA)如何阻止细胞凋亡。

NF-κB controls gene expression of a number of genes and is recently found to be involved in suppression of apoptosis. NF-κB exerts its biological actions through interacting with other cellular proteins. In order to identify molecular partners, we have applied the yeast two-hybrid screening with portions of NF-κB p65 subunit (RelA) as "baits". We have isolated a new gene, called RelA-associated inhibitor (RAI), which have subsequently shown to be located in the nucleus and block the NF-κB -mediated gene expression by inhibiting its DNA binding. Thus, RAI appears to act as a fail-safe mechanism to ensure the silencing of NF-κB or as a feed-back system to downregulate NF-κB activity. Using a different portion of p65 as a "bait", we have identified AES and TLE1, that are the members of Groucho family proteins known as transcriptional co-repressors of Drosophila homologue of human RelA, Dorsal. We have demonstrated that human AES/TLE1 also blocks NF-κB -mediated transcription Since NF-κB has recently been found to be involved in the morphogenesis of higher multicellular organisms, it is expected that human Groucho homologue may be involved in the human body plan in association with NF-κB although the exact mechanism and dynamics should further be explored. Finally, we have identified 53BP2, initially identified as an interacting molecular partner of a tumor suppressor p53 protein, as an interacting protein with p65. When 53BP2 is transduced into cells, these cells were killed with the characteristic features suggesting apoptosis such as fragmented and condensed nuclei and annexin V staining. interestingly, when p65 gene is cotransfected with 53BP2, the 53BP2-induced cell death was completely blocked. These findings suggest that interaction of p65 with 53BP2 may explain, at least in a part, how NF-κB and p65 (RelA) blocks apoptosis.