Phosphorylation is a common type of post-translational modification(PTM).It plays a vital role in many cellular processes.The reversible phosphorylation and dephosphorylation affect protein structures and protein-prot...Phosphorylation is a common type of post-translational modification(PTM).It plays a vital role in many cellular processes.The reversible phosphorylation and dephosphorylation affect protein structures and protein-protein interactions.Previously,we obtained five proteins that interact with ethylene-responsive factor(ERF)from the cDNA library of Populus simonii x Populus nigra.To further investigate the effect of dephosphorylation of PsnERF on its protein binding ability,we generated different phosphorylation states of PsnERF and demonstrated their protein binding capacity by the yeast two-hybrid assay(Y2H).The secondary structures and 3D structures of PsnERF,ERFm,TrunERF,and psnerf^(197/198/202a) were predicted by homology modeling.The Y2H assay indicated that the deletion of serine-rich regions does not affect the interactions,while dephosphorylated mutations blocked the interactions.Homology modeling results suggested that the protein-binding activity was affected by dephosphorylation,and the S197/S198/S202 residues of PsnERF may be the key phosphorylation sites influencing its binding ability.展开更多
Hypertrophic cardiomyopathy (HCM) is a myocardial disorder, with complications including heart failure, thromboemboli and sudden death. Human and feline HCM (fHCM) are clinically comparable, thus fHCM may serve as a s...Hypertrophic cardiomyopathy (HCM) is a myocardial disorder, with complications including heart failure, thromboemboli and sudden death. Human and feline HCM (fHCM) are clinically comparable, thus fHCM may serve as a spontaneous animal model. fHCM in Maine Coon (MC) cats is associated with the p.A31P mutation in the cMyBP-C protein. The mutation is located in the cMyBP-C C0-domain which is known to interact with actin. The presence and levels of the wild type and mutated protein in heart tissue from mutant and wild type MC cats were examined by SDS-PAGE and mass spectrometry (MS). Quantitative yeast-2-hybrid (Y2H) protein-protein interaction analysis was used to assess the effect of the mutation on C0C1/actin interaction. The NMR-based structure of the C0 domain was used to calculate the energetic consequence of replacing alanine with a proline residue. In the homozygous MC cat, the mutated cMyBP-C protein was present, and cMyBPC-C levels were not reduced compared to that of the wild type cat. However, the interaction of actin with mutant cMyBP-C C0C1 was reduced compared to that of wild type. This may be because the substitution of the alanine with proline in position 31 was energetically highly unfavorable and resulted in only one hydrogen bond within the anti-parallel beta-strand compared to two hydrogen-bonds for alanine, possibly destabilizing the structure of the actin-interacting domain. The p.A31P mutation is present in cardiac tissue and the most likely pathogenic mechanism is interference with contractility by reducing binding of the C0C1 domain of cMyBP-C to actin.展开更多
为研究钙依赖蛋白激酶CPK11参与ABA信号通路的方式,本文利用体外酵母双杂实验(Y2H)以及体内双分子荧光互补实验(BiFC)分析CPK11与ABA响应元件结合因子(ABA-responsive element binding factors, ABFs)ABF4之间的关系.酵母双杂交实验表明...为研究钙依赖蛋白激酶CPK11参与ABA信号通路的方式,本文利用体外酵母双杂实验(Y2H)以及体内双分子荧光互补实验(BiFC)分析CPK11与ABA响应元件结合因子(ABA-responsive element binding factors, ABFs)ABF4之间的关系.酵母双杂交实验表明CPK11与ABF4存在体外相互作用,双分子荧光互补实验表明CPK11与ABF4存在体内相互作用.以上实验共同证明CPK11与ABF4存在直接相互作用.作为CPK11的同源蛋白CPK4与转录因子ABF4在植物体内也存在相互作用.以上实验表明CPK11及其同源蛋白CPK4可能通过与转录因子ABF4相互作用从而参与钙离子介导的ABA信号通路.展开更多
基金the Natural Science Foundation of Heilongjiang Province of China(LH2019C059).
文摘Phosphorylation is a common type of post-translational modification(PTM).It plays a vital role in many cellular processes.The reversible phosphorylation and dephosphorylation affect protein structures and protein-protein interactions.Previously,we obtained five proteins that interact with ethylene-responsive factor(ERF)from the cDNA library of Populus simonii x Populus nigra.To further investigate the effect of dephosphorylation of PsnERF on its protein binding ability,we generated different phosphorylation states of PsnERF and demonstrated their protein binding capacity by the yeast two-hybrid assay(Y2H).The secondary structures and 3D structures of PsnERF,ERFm,TrunERF,and psnerf^(197/198/202a) were predicted by homology modeling.The Y2H assay indicated that the deletion of serine-rich regions does not affect the interactions,while dephosphorylated mutations blocked the interactions.Homology modeling results suggested that the protein-binding activity was affected by dephosphorylation,and the S197/S198/S202 residues of PsnERF may be the key phosphorylation sites influencing its binding ability.
文摘Hypertrophic cardiomyopathy (HCM) is a myocardial disorder, with complications including heart failure, thromboemboli and sudden death. Human and feline HCM (fHCM) are clinically comparable, thus fHCM may serve as a spontaneous animal model. fHCM in Maine Coon (MC) cats is associated with the p.A31P mutation in the cMyBP-C protein. The mutation is located in the cMyBP-C C0-domain which is known to interact with actin. The presence and levels of the wild type and mutated protein in heart tissue from mutant and wild type MC cats were examined by SDS-PAGE and mass spectrometry (MS). Quantitative yeast-2-hybrid (Y2H) protein-protein interaction analysis was used to assess the effect of the mutation on C0C1/actin interaction. The NMR-based structure of the C0 domain was used to calculate the energetic consequence of replacing alanine with a proline residue. In the homozygous MC cat, the mutated cMyBP-C protein was present, and cMyBPC-C levels were not reduced compared to that of the wild type cat. However, the interaction of actin with mutant cMyBP-C C0C1 was reduced compared to that of wild type. This may be because the substitution of the alanine with proline in position 31 was energetically highly unfavorable and resulted in only one hydrogen bond within the anti-parallel beta-strand compared to two hydrogen-bonds for alanine, possibly destabilizing the structure of the actin-interacting domain. The p.A31P mutation is present in cardiac tissue and the most likely pathogenic mechanism is interference with contractility by reducing binding of the C0C1 domain of cMyBP-C to actin.
文摘为研究钙依赖蛋白激酶CPK11参与ABA信号通路的方式,本文利用体外酵母双杂实验(Y2H)以及体内双分子荧光互补实验(BiFC)分析CPK11与ABA响应元件结合因子(ABA-responsive element binding factors, ABFs)ABF4之间的关系.酵母双杂交实验表明CPK11与ABF4存在体外相互作用,双分子荧光互补实验表明CPK11与ABF4存在体内相互作用.以上实验共同证明CPK11与ABF4存在直接相互作用.作为CPK11的同源蛋白CPK4与转录因子ABF4在植物体内也存在相互作用.以上实验表明CPK11及其同源蛋白CPK4可能通过与转录因子ABF4相互作用从而参与钙离子介导的ABA信号通路.
