Isolation and identification of NR4A1 interacting with Smac/DIABLO

ZHANG Cui-li; YANG Xiao-ming; CHENG Hai-en; LIU Tao; WANG Ying-xiong

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Isolation and identification of NR4A1 interacting with Smac/DIABLO

更新时间：2025-01-15

- Isolation and identification of NR4A1 interacting with Smac/DIABLO
- Journal of Army Medical University Vol. 29, Issue 6, Pages: 482-485(2007)
- 作者机构：
  
  1. 重庆医科大学：公共卫生学院生殖医学系遗传学教研室，基础医学院生物化学与分子生物学教研室
  2. 军事医学科学院放射医学研究所
  3. 第三军医大学西南医院全军感染病研究所
- 作者简介：
- 基金信息：
- DOI：
  CLC： Q51;Q503;Q785
- Published：2007，
  
  Published Online：30 March 2007，
- 稿件说明：
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ZHANG CUI-LI, YANG XIAO-MING, CHENG HAI-EN, et al. Isolation and identification of NR4A1 interacting with Smac/DIABLO. [J]. Journal of army medical university, 2007, 29(6): 482-485.
DOI：

ZHANG CUI-LI, YANG XIAO-MING, CHENG HAI-EN, et al. Isolation and identification of NR4A1 interacting with Smac/DIABLO. [J]. Journal of army medical university, 2007, 29(6): 482-485. DOI：

摘要

目的利用酵母双杂交技术筛选线粒体促凋亡因子野生型Smac/DIABLO的相互作用蛋白。方法 ①设计促凋亡因子野生型Smac/DIABLO引物，利用PCR在成人肝cDNA文库中扩增野生型Smac/DIABLO基因片段，构建酵母真核表达载体pDBLeu-Smac/DIABLO。②pDBLeu-Smac/DIABLO诱饵质粒转化酵母MaV203

Western blot检测融合蛋白在酵母中的正确表达，自激活作用的检测并确定抑制His基础表达的3AT浓度。③对成人肝cDNA文库的筛选。选择能同时激活His、Ura、LacZ 3个报告基因的克隆为阳性，利用回转实验和GST-pull down 对阳性克隆进行鉴定。结果成功构建pDBLeu-Smac/DIABLO载体，转化酵母细胞MaV203，无细胞毒性，无自激活作用，3AT浓度为25 mmol/L。经回转实验和GST-pull down证实了线粒体促凋亡因子Smac/DIABLO与核受体NR4A1之间具有相互作用。结论 Smac/DIABLO可能通过与核受体NR4A1的相互作用来影响核受体NR4A1对细胞生存或者凋亡的调控。

Abstract

Objective To screen proteins in human adult liver cDNA library interacting with Smac/DIABLO with yeast two-hybrid technique. Methods The wild Smac/DIABLO was amplified with PCR in human adult liver cDNA library. The recombinant vector pDBLeu-Smac/DIABLO was constructed and transformed into yeast MaV203

and the expression of GAL4 BD-Smac/DIABLO fusion protein was dectected by Western blotting. The self-activation was examined

and 3-Amino-1

4-Triazole (3AT) concentration required to titrate basal HIS expression levels was determined. The human adult liver cDNA library was screened sequentially. The clones that grew on SC-Leu-Trp-His-Ura plates was employed to test if they could activate His

Ura and LacZ report genes. The plasmids from those clones that could activate the 3 report genes were isolated

then cotransformed with pDBLeu-Smac/DIABLO

and verified with GST-pull down after sequencing. Results The bait vector pDBLeu-Smac/DIABLO was successfully constructed and expressed correctly in MaV203. One clone nuclear receptor subfamily 4

group A

member 1 (NR4A1) was obtained and could interact with wild Smac/DIABLO via cotransformation and GST-pull down. Conclusion Smac/DIABLO may interact with NR4A1 and affect its function on cell growth and apoptosis.

关键词

Smac/DIABLONR4A1酵母双杂交

Keywords

Smac/DIABLONR4A1yeast two-hybrid

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