Shi Jian-Hong, Zheng Bin, Meng Fang, Wen Jin-Kun, Han Mei
Hebei Laboratory of Medical Biotechnology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang 050017, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2007 Aug;23(3):370-4.
To investigate the interaction between C-terminal domains of SM22alpha and cytoskeleton F-actin.
Prokaryotic expression vector containing SM22alpha cDNA and GST sequence was constructed. The induction conditions were optimized to increase the product of soluble GST-SM22alpha fusion protein in E coli. Expression products were purified and rabbit anti-GST-SM22alpha polyclonal antibody was produced by the purified fusion protein. In order to explore the effect of SM22alpha on cytoskeleton reorganization, VSMCs were treated with serum withdrawal and then serum stimulation to induce contractile/synthetic phenotypic modulation. SM22alpha protein distribution in F-actin/G-actin fractions was detected by Western blotting. The interaction between SM22alpha and actin was examined by GST pull down assay and coimmunoprecipitation. Colocalization of endogenous SM22alpha with F-actin was observed by immunofluorescence.
The results showed that the expression of soluble GST-SM22alpha protein was the highest under condition induced by 30 degrees C, 0.5 mmol/L IPTG for 6 h. Immunofluorescence and Western blotting of protein extracts from F-actin/G-actin fractions revealed that SM22alpha colocalized with F-actin during VSMC redifferentiation. GST pull down assay and coimmunoprecipitation showed that SM22alpha interacted with F-actin by C-terminal domains to participate in cytoskeleton reorganization.
The recombinant SM22alpha C-terminal domains have the ability to bind F-actin, by which SM22alpha interacts with actin and participates in cytoskeleton reorganization.
研究平滑肌22α（SM22α）的C末端结构域与细胞骨架F-肌动蛋白之间的相互作用。
构建含SM22α cDNA和谷胱甘肽-S-转移酶（GST）序列的原核表达载体。优化诱导条件以增加大肠杆菌中可溶性GST-SM22α融合蛋白的产量。对表达产物进行纯化，并用纯化的融合蛋白制备兔抗GST-SM22α多克隆抗体。为了探究SM22α对细胞骨架重组的影响，对血管平滑肌细胞（VSMCs）进行血清饥饿处理，然后血清刺激以诱导收缩/合成表型调节。通过蛋白质免疫印迹法检测F-肌动蛋白/G-肌动蛋白组分中SM22α蛋白的分布。通过GST下拉实验和免疫共沉淀检测SM22α与肌动蛋白之间的相互作用。通过免疫荧光观察内源性SM22α与F-肌动蛋白的共定位。
结果表明，在30℃、0.5 mmol/L异丙基-β-D-硫代半乳糖苷（IPTG）诱导6小时的条件下，可溶性GST-SM22α蛋白的表达量最高。对F-肌动蛋白/G-肌动蛋白组分的蛋白质提取物进行免疫荧光和蛋白质免疫印迹分析显示，在VSMC再分化过程中，SM22α与F-肌动蛋白共定位。GST下拉实验和免疫共沉淀表明，SM22α通过C末端结构域与F-肌动蛋白相互作用，参与细胞骨架重组。
重组SM22α C末端结构域具有结合F-肌动蛋白的能力，通过该能力SM22α与肌动蛋白相互作用并参与细胞骨架重组。
