The tyrosine kinase receptor vascular endothelial growth factor receptor 2 （VEG FR2） is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues formi...The tyrosine kinase receptor vascular endothelial growth factor receptor 2 （VEG FR2） is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetyiases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In par- ticular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phos- phoryiation and intraceUular signaling upon proLonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetyiation is a critical mechanism that directLy affects VEGFR2 function.展开更多
RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues.Injectable hydrogels are one potentially simple and dire...RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues.Injectable hydrogels are one potentially simple and direct route towards overcoming these barriers.Here we report on the utility of a combination of a mildly modified form of the clinically utilised fibrin hydrogel with Invivofectamine^(■)3.0,a lipid nonviral transfection vector,for local and sustained release.PEGylation of fibrin allowed for controlled release of small interfering RNA(siRNA)-lipopolyplexes for at least 10 days and greatly increased the stability of fibrin in vitro and in vivo.A 3D cell culture model and a release study showed transfection efficacy of siRNA-lipopolyplexes was retained for a minimum of 7 days.Injection in conjunction with PEGylated-fibrinogen significantly increased retention of siRNA-lipopolyplexes in mouse skeletal muscle and enhanced knockdown of myostatin mRNA that correlated with muscle growth.Thus,the increased efficacy observed here for the combination of a lipid nanoparticle,the only type of nonviral vector approved for the clinic,with fibrin,might allow for more rapid translation of injectable hydrogel-based RNA interference.展开更多
文摘The tyrosine kinase receptor vascular endothelial growth factor receptor 2 （VEG FR2） is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetyiases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In par- ticular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phos- phoryiation and intraceUular signaling upon proLonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetyiation is a critical mechanism that directLy affects VEGFR2 function.
基金supported by the National Research Foundation of South Africa(93550)International Centre for Genetic Engineering and Biotechnology(CRP/ZAF14-01)the South African Medical Research Council(416007).
文摘RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues.Injectable hydrogels are one potentially simple and direct route towards overcoming these barriers.Here we report on the utility of a combination of a mildly modified form of the clinically utilised fibrin hydrogel with Invivofectamine^(■)3.0,a lipid nonviral transfection vector,for local and sustained release.PEGylation of fibrin allowed for controlled release of small interfering RNA(siRNA)-lipopolyplexes for at least 10 days and greatly increased the stability of fibrin in vitro and in vivo.A 3D cell culture model and a release study showed transfection efficacy of siRNA-lipopolyplexes was retained for a minimum of 7 days.Injection in conjunction with PEGylated-fibrinogen significantly increased retention of siRNA-lipopolyplexes in mouse skeletal muscle and enhanced knockdown of myostatin mRNA that correlated with muscle growth.Thus,the increased efficacy observed here for the combination of a lipid nanoparticle,the only type of nonviral vector approved for the clinic,with fibrin,might allow for more rapid translation of injectable hydrogel-based RNA interference.
