The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosollc DNA derived from microbial pathogens or mls-located cellular DNA.Upon DNA binding,cGAS utilizes GTP and ATP as subs...The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosollc DNA derived from microbial pathogens or mls-located cellular DNA.Upon DNA binding,cGAS utilizes GTP and ATP as substrates to synthesize cGAMP,leading to MITA-mediated innate immune response.In this study,we identified the phosphatase PPP6C as a negative regulator of cGASmediated innate immune response.PPP6C is constitutively associated with cGAS in un-stimulated cells.DNA virus infection causes rapid disassociation of PPP6C from cGAS,resulting in phosphorylation of human cGAS S435 or mouse cGAS S420 in its catalytic pocket.Mutation of this eerine residue of cGAS impairs its ability to synthesize cGAMP upon DNA virus infection.In vitro experiments indicate that S420-phosphorylated mcGAS has higher affinity to GTP and enzymatic activity.PPP6Cdeficiency promotes innate immune response to DNA virus in various cells.Our findings suggest that PPP6Cmediated dephosphorylation of a catalytic pocket serine residue of cGAS impairs its substrate binding activity and innate immune response,which provides a mechanism for keeping the DNA sensor cGAS inactive in the absence of infection to avoid autoimmune response.展开更多
Synthesis of chiral amines by amine dehydrogenase has the advantages of environmental friendliness,high stereoselectivity,and mild reaction conditions.However,amine dehydrogenase has low catalytic activity,which great...Synthesis of chiral amines by amine dehydrogenase has the advantages of environmental friendliness,high stereoselectivity,and mild reaction conditions.However,amine dehydrogenase has low catalytic activity,which greatly limits its application in the large-scale synthesis of chiral amines.In this study,a novel amine dehydrogenase was obtained by modifying the substrate specificity of leucine dehydrogenase via computer-aided protein engineering strategy.Furthermore,conservation analysis,homology modeling and molecular docking analysis were carried out via biocomputing strategy to select the mutation sites,and the mutants L52S and T143C were obtained.The enzyme activities of the two mutants to 2-pentanone were 1.55 U/mg and 2.06 U/mg,respectively.The enzyme activity of the latter was 188%and the Tm value was 2.55°Chigher than those of the original mutant,which laid a foundation for the efficient preparation of chiral amines by using this novel enzyme.展开更多
基金This work was supported by grants from the State Key R&D Program of China(2017YFA0505800,2016YFA0502102)the National Natural Science Foundation of China(Grant Nos.31830024 and 31630045).
文摘The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosollc DNA derived from microbial pathogens or mls-located cellular DNA.Upon DNA binding,cGAS utilizes GTP and ATP as substrates to synthesize cGAMP,leading to MITA-mediated innate immune response.In this study,we identified the phosphatase PPP6C as a negative regulator of cGASmediated innate immune response.PPP6C is constitutively associated with cGAS in un-stimulated cells.DNA virus infection causes rapid disassociation of PPP6C from cGAS,resulting in phosphorylation of human cGAS S435 or mouse cGAS S420 in its catalytic pocket.Mutation of this eerine residue of cGAS impairs its ability to synthesize cGAMP upon DNA virus infection.In vitro experiments indicate that S420-phosphorylated mcGAS has higher affinity to GTP and enzymatic activity.PPP6Cdeficiency promotes innate immune response to DNA virus in various cells.Our findings suggest that PPP6Cmediated dephosphorylation of a catalytic pocket serine residue of cGAS impairs its substrate binding activity and innate immune response,which provides a mechanism for keeping the DNA sensor cGAS inactive in the absence of infection to avoid autoimmune response.
基金supported by the Key Technology Research Plan Project of the Inner Mongolia Autonomous Region(2019GG302)the Program of the Key Laboratory of Carbohydrate Chemistry&Biotechnology,Ministry of Education,China(KLCCB-KF202103).
文摘Synthesis of chiral amines by amine dehydrogenase has the advantages of environmental friendliness,high stereoselectivity,and mild reaction conditions.However,amine dehydrogenase has low catalytic activity,which greatly limits its application in the large-scale synthesis of chiral amines.In this study,a novel amine dehydrogenase was obtained by modifying the substrate specificity of leucine dehydrogenase via computer-aided protein engineering strategy.Furthermore,conservation analysis,homology modeling and molecular docking analysis were carried out via biocomputing strategy to select the mutation sites,and the mutants L52S and T143C were obtained.The enzyme activities of the two mutants to 2-pentanone were 1.55 U/mg and 2.06 U/mg,respectively.The enzyme activity of the latter was 188%and the Tm value was 2.55°Chigher than those of the original mutant,which laid a foundation for the efficient preparation of chiral amines by using this novel enzyme.
