紫外辐射诱导的细胞凋亡中PUMA与Bcl-2家族蛋白间调控的分子成像研究

PUMA Promotes Bax Translocation by Competitive Binding to Bcl-Xl during UV Irradiation-Induced Apoptosis

紫外辐射诱导的细胞凋亡是一个包括DNA损伤、死亡受体启动、线粒体破裂等一系列反应的复杂过程。有研究报道,由紫外辐射引发的DNA损伤可以激活肿瘤抑制因子p53,从而触发细胞凋亡级联反应,其中一个重要的事件就是上调促凋亡蛋白(p53 up-regulated modulator of apoptosis,PUMA)的表达。但是,PUMA在紫外辐射诱导凋亡过程中的作用机制尚不清楚。利用荧光共振能量转移技术(Fluorescence resonance energy transfer,FRET),在人类肺腺癌细胞(ASTC-α-1)内实时研究了紫外辐射诱导凋亡过程中PUMA,Bax,Bcl-Xl三者之间的相互作用关系,从而揭示PUMA的作用机制。用120 mJ/cm2的紫外辐射剂量诱导细胞凋亡,通过共转染荧光探针GFP-PUMA和YFP-Bcl-Xl或者CFP-Bcl-Xl和YFP-Bax,在激光共聚焦扫描显微镜下实时检测PUMA和Bcl-Xl或者Bcl-Xl和Bax之间的相互作用。结果显示,紫外辐射后GFP-PUMA与YFP-Bcl-Xl之间的FRET现象逐渐增强,说明PUMA与Bcl-Xl的作用越来越强;CFP-Bcl-Xl与YFP-Bax之间的FRET现象逐渐减弱,说明Bcl-Xl与Bax的作用越来越弱,此过程伴随有YFP-Bax在线粒体的明显定位。该实验结果暗示PUMA是竞争结合了Bcl-Xl,解除了Bcl-Xl对Bax的抑制作用,进而活化了Bax。

UV-mediated apoptosis is a highly complex process in which different molecular pathways are involved. These include DNA damage,triggering of cell death receptors and the broken of mitochondria. It has been reported that DNA damage which is induced by UV irradiation can active the tumor suppressor p53 and trigger a cascade response of cell apoptosis. One of the most importment thing is the expression of PUMA is upregulated. However,the mechanism of PUMA-mediated apoptosis and Bax translocation after UV irradiation remains unclear. In this study,the fluorescence resonance energy transfer （FRET） technique was used to study the dynamic interaction among PUMA,Bax and Bcl-Xl in living single-cells. ASTC-a-1 cells were transiently co-transfected with GFP-PUMA and YFP-Bcl-Xl or CFP-Bcl-Xl and YFP-Bax,followed with 120 mJ/cm^2 UV irradiation. We found that the FRET intensity of GFP-PUMA and YFP-Bcl-Xl increased while the FRET intensity of CFP-Bcl-Xl and YFP-Bax dicreased suggesting PUMA has a more strong interaction with Bcl-Xl than Bax. At the same time,GFP-Bax translocated to the mitochondria from the cytosol in typical cells started at about 7 h after UV irradiation. Taken together,the results indicated that PUMA promotes Bax translocation by competitive binding to Bcl-Xl during UV-induced apoptosis.