| Microvascular angiogenesis is a budding way to grow and form new blood vessels from the existing vascular bed. This process is the result of proliferation, migration and remodeling of the vascular endothelial cells. It concludes the following five stages:①Degradation of the vascular basement membrane by protease;②Endothelial cells pass through the basement membrane around blood vessels and then migrate to the matrix;③Endothelial cells proliferation, adhesion and connection;④Endothelial cells form the tube-like structure;⑤Matrix remodeling, smooth muscle cells wrapping, vascular anastomosis and vascular network formation.Among the anti-cancer drugs currently being developed, one is to direct for the process of tumor angiogenesis, which is to achieve tumor suppressed by this inhibitory effects. In order to continue to provide nutrition for growing when solid tumors grow to 2-3 mm3, they can form a large number of new blood vessels, which need an effective mechanism of angiogenesis. Inhibition the function of endothelial cells may show an effective reduction in growth of the tumors, and such a starvation diet will be a typical effective means of cancer treating.In recent years, studies have found that endothelial monocyte-activating polypeptideⅡ(EMAPⅡ) is an effective angiogenesis inhibitor and an active cytokine. Later found that its precursor protein, pro-EMAPⅡ, is also an effective angiogenesis inhibitor, which have shown inhibition of angiogenesis in vitro and in vivo, and is expected to be a candidate of drugs to treat solid tumors.pro-EMAPⅡprotein, also known as p43 protein, is an auxiliary factor of mammalian multi-aminoacyl-tRNA synthetases, with 312 amino acids, molecular weight of 34 kD. In certain conditions, the digestion site ASTD motif of pro-EMAPⅡprotein can be identified specifically by caspase-7 and pro-EMAPⅡprotein can be cut into two domains after the aspartic acid at position 146, and the C-terminal domain is EMAPⅡprotein.If the aspartic acid of the ASTD sequence in pro-EMAPⅡprotein is replaced by an alanine, caspase-7 will not recognize the ASTA motif and pro-EMAPⅡprotein can not be cut into two domains. Studies on the differences of the activities between pro-EMAPⅡprotein and its mutant will be capable to reveal the mechanism of pro-EMAPⅡprotein playing the biological activities.Reported in the literature that tumor angiogenesis-related endothelial cell receptor mainly includes several categories in the following: vascular endothelial growth factor receptor, Tie receptors, intergrins and theαsubunit of ATP synthase and so on. The receptor of angiostatin in endothelial cell surface is theα/βsubunit of ATP synthase. The research shows that, EMAPⅡprotein also has interaction with theαsubunit of ATP synthase in the cell surface.Theαsubunit of ATP synthase, also known as ATP5A protein, is part of the hydrophilic catalytic core F1 complex outside the membrane of the whole enzyme and acts as a catalyst for ATP synthesis, which consists of 553 amino acids, molecular weight of 60 kD. In the past ATP synthase was considered a strict mitochondrial protein, recent studies have shown that some subunits of the enzyme are widely distributed in the plasma membrane surface of a number of tumor cells and endothelial cells, known as the ectopic ATP synthases to plasma membrane. These ATP synthases involved in various biological processes as receptors of a variety of ligands.The research of interaction between ATP5A protein and pro-EMAPⅡprotein will help to reveal the mechanisms of pro-EMAPⅡprotein inhibiting angiogenesis and is of great importance to the studies of its receptor and signal pathways.Firstly this paper is to study the differences of activities in inhibiting angiogenesis by mutation in protease cleavage site of pro-EMAPⅡ.In the experiment the mutant gene was obtained by overlapping PCR, which was mutation of aspartate at position 146 into alanine. The mutant protein was expressed as a fusion protein containing 6×His tag in E.coli, was purified by SP ion exchange chromatography and Ni affinity chromatography, and the purity was more than 90%. We studied on the differences of angiogenesis inhibition between pro-EMAPⅡprotein and its mutant by the tube formation assay and cell migration assay of HUVEC cells, the results show that the activities increased after mutation of pro-EMAPⅡprotein, indicating that the inhibiting neovascularization capacity was improved after mutation of pro-EMAPⅡprotein.The inhibition of neovascularization capacity of pro-EMAPⅡprotein increased when the aspartic acid at position 146 was replaced by alanine in the mutant, indicating that the aspartic acid at position 146 in this protein plays a key role. The mutation may make the tertiary structure of pro-EMAPⅡprotein becoming looser, so the activities of inhibiting angiogenesis of EMAPⅡprotein are part of released and maybe the auxiliary functions of the N-terminal domain are released too, in the result of inhibiting angiogenesis capacity of pro-EMAPⅡmutant increased. The other groups of our laboratory found, the inhibition of neovascularization capacity of separate N-terminal domain and the C-terminal domain (EMAPⅡ) weakened compared to pro-EMAPⅡprotein, and the N-terminal domain was weaker than the C-terminal domain, but the inhibition of neovascularization capacity of the N-terminal domain and the C-terminal domain simultaneously was significantly higher than pro-EMAPⅡprotein. These results indicate that the most likely mechanism of inhibiting angiogenesis of pro-EMAPⅡprotein is that pro-EMAPⅡprotein was digested with the release of N-terminal domain and the C-terminal domain, and C-terminal domain plays angiogenesis inhibition, while the N-terminal domain plays a supporting role, and then the two domains function together to achieve the effects of inhibition of angiogenesis.Then we have a preliminary study on the interaction between pro-EMAPⅡprotein and ATP5A protein in vitro. First HepG2 cells were identified as cell line using by immunofluorescence experiments, and then its total RNA was extracted. ATP5A gene was obtained by RT-PCR cloning. ATP5A protein was expressed as a fusion protein containing 6×His tag in E.coli with prokaryotic expression system. It was expressed as inclusion body by anlysis and was purified by SP ion exchange chromatography and Ni affinity chromatography after denatured, followed by dialysis, western blotting. Results revealed ATP5A protein refolded whose conformation consistent with the natural protein was obtained with purity of 85%. At last we found that pro-EMAPⅡprotein and ATP5A protein had a certain binding effect in vitro by ELISA and immunoprecipitation experiments.In summary, the studies on the mutation of cleavage site in pro-EMAPⅡis helpful to reveal the mechanism of pro-EMAPⅡprotein playing roles in inhibiting angiogenesis; the studies of the interaction between pro-EMAPⅡprotein and ATP5A protein confirm that pro-EMAPⅡprotein and ATP5A protein have existed a certain binding effect in vitro, which is the basis for the study of pro-EMAPⅡprotein-related signaling pathway.
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