Recombination And Expression Of HSG And Its Deleted Mutants And Study Of Their Functions

Hyperplasia suppressor gene ( HSG,also named mitofusin-2) was isolated by screening vascular smooth muscle cell (VSMC) cDNA library of Wistar-Kyoto rats (WKY) and spontaneous hypertension rats (SHR) using differential display method. We previously reported that overexpression of HSG gene decreased the expression of cyclin E protein and proliferation of VSMCs by inhibition of the Ras–Raf–MEK–ERK1/2 signaling pathway. The results of coimmunoprecipitation and confocal microscopy assay showed that HSG could associate with SMα-actin in cytoplasm of VSMCs. These findings implied that HSG participate in VSMC cytoskeleton reorganization and differentiation by interacting with actin. HSG encodes a protein of 757 amino acids. The protein contains several domains including a hydrophobic transmembrane domain (amino acids 599–644), a p21ras signature motif (amino acids 77–92), a GTP-binding site motif (P-loop; amino acids 98–117), and a possible PKA/PKG phosphorylation site at Ser 442,which have different functions. It is still unclear for the domain interacted with actin, and the biological significance of HSG-actin interaction is poorly understood. To study the structural domain of interaction between HSG and SM-αactin and explore the mechanisms whereby HSG regulates cytoskeleton reorganization.HSG and its deleted mutants were constructed and F-actin polymerization and cross-linking assays in vitro were performed. In addition, we also constructed a recombinant adenovirus expression vector of the human HSG and made it expressed in VSMC efficiently.Methods:1 Construction of recombinant plasmids HSG and its deleted mutants cDNA fragments were amplified by PCR and cloned into the prokaryotic expression vector pGEX-3X after digested with BamH I / EcoR I. The recombinant plasmids which containing different HSG named as pGEX-3X-HSG(323- 2596bp), pGEX-3X-HSG-1(323-1441bp), pGEX-3X-HSG-2(1562-2116bp)and pGEX-3X-HSG-3(1562- 2596bp), respectively.2 Expression and Purification of GST-HSG fusion protein2.1 Expression of GST-HSG fusion proteinThe E. coli transformed by pGEX-3X-HSG-1(323-1441bp), pGEX-3X-HSG-2(1562-2116 bp) and pGEX-3X-HSG-3(1562- 2596bp) respectively was induced by different IPTG concentration, time and temperature to identify the optimal induction condition. Supernatant and precipitation of the lysates were analyzed by SDS-PAGE.2.2 Purification of GST-HSG fusion proteinAfter inclusion bodies were cleaned for several times, they were further denatured by sarcosyl and then renatured by means of dialysis. Renatured fusion proteins were purified via GST-Sepharose 4B affinity resin.3 Structure domain of interactin between HSG and actin Purified GST-HSG1,GST-HSG2 fusion protein and F-actin were incubated together,then centrifuged to separate cross-linked F-actin. Protein in the pellets and supernatants were analyzed by Western blotting.4 Effects of HSG on cytoskeleton reorganization in VSMCs VSMCs were treated with serum and then withdrawed to induce synthetic/contractile phenotype remodulation. Distributions of HSG and actin in F-actin/G-actin fractions were detected by step-by-step extraction of proteins from VSMCs and Western blotting.Results:1 Construction of recombinant plasmidsThe analysis of DNA sequence and restriction enzyme analysis showed that the inserted fragments of pGEX-3X-HSG (323-2596bp),pGEX-3X-HSG-1(323-1441bp), pGEX-3X-HSG -2(1562-2116bp) and pGEX-3X-HSG-3(1562-2596bp) were consistent with the reported sequence.2 Expression of GST-HSG fusion proteinGST-HSG1,GST-HSG2 and GST-HSG3 fusion proteins could be expressed in E. coli transformed by pGEX-3X-HSG-1 (323-1441bp), pGEX-3X-HSG-2(1562-2116bp) and pGEX-3X- HSG-3(1562-2596bp),respectively as inclusion body after induction. The expression of these fusion proteins was the highest under condition induced by 0.1 mmol/L IPTG for 6 h at 33℃.3 Purification of GST-HSG fusion proteinAfter inclusion bodies were cleaned for several times, renatured fusion proteins were purified via GST-Sepharose 4B affinity resin. 5 mg of purified GST-HSG fusion protein was obtained in 100 ml culture, the purity of GST-HSG fusion protein was over 90 %.4 After GST-HSG1 and GST-HSG2 fusion protein were incubated together with F-actin at the ratio of 1:1, the polymerized F-actin was not detected by Western blotting, suggesting that GST-HSG1 and GST-HSG2 did not promote polymerization and cross-linking of actin. However,with the increasing of the ratio of GST-HSG/actin and GST-HSG1, but not GST-HSG2,increased in cross-linked F-actin level.5 Serum starvation could induce the expression of HSG protein, but the distribution of HSG in F-actin and G-actin was not different after serum starvation.Conclusions:1 Prokaryotic expression plasmids namely pGEX-3X-HSG pGEX-3X-HSG-1, pGEX-3X-HSG-2,and pGEX-3X-HSG-3 are successfully constructed.2 GST-HSG1,GST-HSG2 and GST-HSG3 fusion proteins are effectively expressed in E.coli under condition induced by 0.1 mmol/L IPTG for 6 h at 33℃. 3 GST-HSG1,GST-HSG2 and GST-HSG3 fusion proteins are purified via GST-Sepharose 4B affinity resin, and the purity of fusion protein was reached to over 90 %.4 GST-HSG1 participated in promoting the cross-linking of F-actin. HSG-actin interaction was affected by the ratio of GST-HSG/actin.