IngentaConnect Fluorescence Resonance Energy Transfer Analysis of Bid Activation...

Authors: WU, Yinyuan¹; XING, Da; LIU, Lei¹; CHEN, Tongsheng¹; CHEN, Wei R.¹

Source: Acta Biochimica et Biophysica Sinica, Volume 39, Number 1, January 2007 , pp. 37-45(9)

Abstract:

Ultraviolet (UV) irradiation is a DNA-damaging agent that triggers apoptosis through both the membrane death receptor and mitochondrial apoptotic signaling pathways. Bid, a pro-apoptotic Bcl-2 family member, is important in most cell types to apoptosis in response to DNA damage. In this study, a recombinant plasmid, YFP-Bid-CFP, comprised of yellow and cyan fluorescent protein and a full length Bid, was used as a fluorescence resonance energy transfer analysis (FRET) probe. Using the FRET technique based on YFP-Bid-CFP, we found that Bid activation was initiated at 9±1 h after UV irradiation, and the average duration of the activation was 75±10 min. Bid activation coincided with a collapse of the mitochondrial membrane potential with an average duration of 50±10 min. When cells were pretreated with Z-IETD-fmk (caspase-8 specific inhibitor) the process of Bid activation was completely inhibited, but the apoptosis was only partially affected. Z-DEVD-fmk (caspase-3 inhibitor) and Z-FA-fmk (non asp specific inhibitor) did not block Bid activation. Furthermore, the endogenous Bid activation with or without Z-IETD-fmk in response to UV irradiation was confirmed by Western blotting. In summary, using the FRET technique, we observed the dynamics of Bid activation during UV-induced apoptosis and found that it was a caspase-8 dependent event.

Edite by Xianxi WANG

Keywords: ultraviolet irradiation; Bid activation; apoptosis; caspase-8; fluorescence resonance energy transfer analysis

Document Type: Research article

DOI: 10.1111/j.1745-7270.2007.00246.x

Affiliations: 1: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China

The full text electronic article is available for purchase. You will be able to download the full text electronic article after payment.

$50.16 plus tax Refund Policy

Fluorescence Resonance Energy Transfer Analysis of Bid Activation in Living Cells during Ultraviolet-induced Apoptosis