神经型一氧化氮合酶(nNOS)与SUMO连接酶PIAS3相互作用结构基础鉴定  

Structural Basis Identification of Interaction between Neuronal Nitric Oxide Synthase and SUMO Ligase PIAS3

作　　者：胡露露 杜彩萍 HU Lu-Lu;DU Cai-Ping(Research Center for Biochemistry and Molecular Biology,Jiangsu Key Laboratory of Brain Disease Bioinformation,Xuzhou Medical University,Xuzhou 221004,Jiangsu,China)

机构地区：[1]徐州医科大学,江苏省脑病生物信息重点实验室,生物化学与分子生物学研究中心,江苏徐州221004

出　　处：《中国生物化学与分子生物学报》2020年第9期1055-1063,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.81100852);江苏省徐州市科技计划项目(No.KC18205)资助。

摘　　要：神经型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)在中枢神经系统广泛分布。本课题组前期研究报道,神经活性增强促进神经型一氧化氮合酶SUMO化,继而上调其活性,激活下游ERK1/2信号通路,参与调控兴奋性突触传递。SUMO连接酶PIAS3可通过其N-末端AAs 43~86与nNOS结合,参与介导神经型一氧化氮合酶SUMO化。本文进一步筛选和鉴定nNOS中与PIAS3结合的氨基酸序列,深入研究nNOS与PIAS3相互作用的结构基础。采用分子克隆技术,分别构建nNOS缺失结构域突变体(AAs 1~1396、1~756和1~720)的真核表达载体,然后分别与Myc-PIAS3质粒共转染于COS7细胞。结果显示,AAs 1~720与PIAS3共转染组nNOS-PIAS3的结合水平显著低于其他组;Myc-nNOS(AAs 721~756)化学合成小肽与纯化的His-PIAS3在体外也可相互结合。结果表明,nNOS的AAs 721~756(CaM结合结构域,CaMB)可与PIAS3直接结合。GST pull-down进一步证明,nNOS的CaMB与PIAS3 AAs 43~86可直接结合。构建CaMB-pcDNA3.1真核表达载体,共表达nNOS、Myc-PIAS3和CaMB,CaMB共表达组nNOS-PIAS3结合明显降低。结果提示,CaMB通过竞争结合PIAS3从而干预nNOS与PIAS3的结合。将化学合成的穿膜肽Tat融合的CaMB(Tat-CaMB,5μmol/L)与皮质神经元预孵育1 h,用荷包牡丹碱(bicuculline,50μmol/L)诱导化学性长时程增强(long-term potentiation,LTP)。结果显示,Tat-CaMB可显著逆转bicuculline诱导的ERK1/2磷酸化(活化)水平的升高,与荷包牡丹碱处理组相比,下降了29%(P<0.05)。结果表明,Tat-CaMB可通过干预nNOS-PIAS3结合,进而抑制神经型一氧化氮合酶SUMO化及其下游ERK1/2信号通路的活化。通过上述研究,证明nNOS可通过CaM结合结构域(AAs 721~756)与PIAS3结合,并提供了一种鉴定和验证蛋白质间相互作用结构基础的有效方法。Neuronal nitric oxide synthase(nNOS)is wildly distributed in the central nervous system.Our previous study reported that the neuronal activity-induced nNOS SUMOylation upregulates its enzymatic activity,then activates downstream ERK1/2 signal cascades,and ultimately participates in the regulation of excitatory synaptic transmission.In addition,SUMO ligase PIAS3 bound to nNOS through its N-terminal amino acids(AAs)43~86,and involved in nNOS SUMOylation.In order to further study the structural basis for the nNOS-PIAS3 interaction,the amino acids in nNOS associated with PIAS3 were screened and identified.Using molecular cloning method,the eukaryotic expression vector of domain-deleted nNOS mutants(AAs 1~1396,1~756 and 1~720)were constructed,and then co-expressed with Myc-tagged PIAS3(Myc-PIAS3)in COS7 cells.The nNOS-PIAS3 interaction in the group co-transfected with AAs 1~720 with PIAS3 was significantly lower than that in other groups.Moreover,small peptides of Myc-nNOS(AAs 721~756)bound to purified His-PIAS3 in vitro.These data suggested that AAs 721~756 in nNOS(CaM binding domain,CaMB)interact with PIAS3 directly.GST pull-down assay further proved that CaMB in nNOS interacts with AAs 43~86 in PIAS3 directly.Next,CaMB-pcDNA3-1 eukaryotic expression vector was constructed,and then cotransfected with nNOS and Myc-PIAS3 expressing plasmids in COS7 cells.The binding between nNOS and PIAS3 was obviously reduced by CaMB,indicating that CaMB competitively combines with PIAS3 to interfere nNOS-PIAS3 association.Then cortical neurons were pre-incubated with 5μmol/L small peptides fused with the cell membrane-penetrating peptide Tat,Tat-tagged nNOS segment representing amino acids 721~756(Tat-CaMB),for 1 h and then treated with 50μmol/L bicuculline to induce chemical long-term potentiation(LTP).Pretreatment with Tat-CaMB significantly reversed the bicuculline-induced elevation of ERK1/2 phosphorylation,with 29%(P<0.05)decrease as compared to bicuculline treated group.The data suggested that Tat-CaMB inhibits nNOS SUMOylat

关 键 词：神经型一氧化氮合酶 SUMO连接酶 PIAS3 CaM结合结构域 结合 

分 类 号：Q189[生物学—神经生物学] Q78[生物学—普通生物学]