活化的T细胞核内因子（NFAT）的调节及其在神经系统中的功能

活化的T细胞核内因子（nuclear factor of activated T-cells, NFAT）作为细胞信号转导通路中的一类重要的转录因子参与细胞功能的调节. NFAT的活化主要是通过细胞内钙/钙调神经磷酸酶（Ca2+/calcineurin）的刺激启动,它脱磷酸后发生核转位并与DNA的特定序列结合,同时通过与其它转录因子的协同作用,调节目的基因的特定表达. NFAT在免疫系统中所调节的基因表达已经得到了充分的研究. 近年实验研究发现,NFAT的转录因子家族在脊椎动物的神经系统中也发挥着非常重要的作用. 本文综述了NFAT家族蛋白的分类、结构、磷酸酶与激酶对其出入核的调节及在神经系统中的研究进展,使得能够更加全面地认识calcineurin/NFAT信号通路的作用. 此外,由于环孢菌素A（cyclosporin A）等药物在神经系统应用的局限性,对于NFAT调节深入研究,也将为筛选或者开发更为高效、低毒药物提供新的思路.     

钙调磷酸酶信号调控真菌生长代谢、毒力及抗逆性能

钙调磷酸酶是一种丝氨酸/苏氨酸（Ser/Thr）蛋白磷酸酶,在真菌中普遍保守,上游信号途径由Ca²⁺通道（Cch1）、转运蛋白（Mid1）、钙离子感应蛋白（CaM）、钙调蛋白依赖性磷酸酶等组成。钙调磷酸酶受钙离子和钙调蛋白调节,在调控真菌Ca²⁺稳态的钙信号级联途径中发挥着中心作用,通过钙信号级联途径参与生物学过程,调控真菌生长、发育和毒力形成来响应外界环境因素的变化,使真菌能够适应不同环境,维持正常的生命活动。本文综述了真菌钙调磷酸酶信号的组成和上下游信号转导途径、调控细胞生长代谢、毒力形成以及抗逆性能调控的研究进展;结合对真菌代谢产物合成的调控作用,对钙调磷酸酶信号作为重要合成生物学元件及调控开关进行了展望。     

果蝇钙调蛋白和肌球蛋白NINAC相互作用分析

果蝇的视觉信号转导途径是已知的最快的G 蛋白偶联信号通路。这其中涉及到TRP/TRPL通道的开放以及钙离子的内流等一系列反应的形成。NINAC（neither inactivation nor afterpotential C）是一种特异性存在于果蝇感光细胞中的第3类肌球蛋白（Myosin III）,其在终止果蝇的视觉信号转导通路中起着非常重要的作用。NINAC蛋白具有两种亚型：一种是132 kD的蛋白亚型 (p132),另一种则是174 kD的蛋白亚型(p174)。这两种不同的蛋白亚型都具有相同的激酶催化结构域(kinase domain),以及与肌球蛋白相似的马达结构域(motor domain)。但是,它们在C末端却存在着非常大的差异,这其中包括了钙调蛋白结合基序(IQ motif)。NINAC的这两种蛋白亚型在果蝇的感光细胞中的定位以及作用有很大不同,尤其是在与钙调蛋白的相互作用方面。钙调蛋白结合基序与钙调蛋白（CaM）之间的相互作用对于果蝇的视觉信号通路具有重要的意义：NINAC结合钙调蛋白能力的缺失将导致果蝇的视觉传导缺陷。本文通过蛋白共表达的方法,成功表达并纯化得到了不同版本的NINAC与钙调蛋白的蛋白复合物。静态光散射的结果表明,在Ca2+存在情况下,p174蛋白可以结合2个Ca2+-CaM,而p132只结合1个Ca2+-CaM。通过分析型凝胶过滤以及等温量热滴定技术,进一步鉴定了p174及p132的IQ2（第2个钙调蛋白结合基序）序列与Ca2+ CaM的相互作用。通过序列分析及进一步的突变实验发现,p174 IQ2中的3个疏水氨基酸（F1083,F1086 和 L1092）对于钙调蛋白的结合非常重要,并导致了p174与p132蛋白和Ca2+ CaM结合能力的差异。本文的研究提供了NINAC与Ca2+-CaM相互作用的生化机制,将为进一步在果蝇视觉信号通路中深入研究CaM是如何调节NINAC的体内功能实验打下基础。     

玉米大斑病菌钙调磷酸酶A亚基的克隆与特征分析

根据已知病原真菌钙调磷酸酶A亚基(Calcineurin A, CNA)丝苏蛋白磷酸酶保守结构域设计引物, 从玉米大斑病菌cDNA中扩增出CNA基因片段。利用cDNA末端快速克隆(Rapid amplification of cDNA ends, RACE)手段获得该基因全长cDNA序列(GenBank登录号: EF 407562)。Southern杂交结果显示, 该基因在玉米大斑病菌基因组只有1个拷贝。CNA特异性抑制剂Cyclosporin A对玉米大斑病菌分生孢子和附着胞发育有抑制作用, 抑制作用与抑制剂浓度呈正相关, 相同浓度的抑制剂对附着胞形成的抑制作用大于对孢子萌发的抑制。经CsA(5µg/mL)处理后, 玉米大斑病菌分生孢子不能侵入玉米叶片, 初步表明CNA基因参与玉米大斑病菌的致病过程。     

灵芝钙调蛋白基因CaM的克隆与功能分析

灵芝是我国传统的食药用真菌,具有广泛的免疫调节功能并含有多种生物活性成分。灵芝酸是灵芝的主要次生代谢产物之一,具有较高商业价值。钙调蛋白（calmodulin,CaM）作为真核生物中重要的Ca ²⁺信使,能够参与生长发育、次生代谢等重要生理过程。本研究通过序列分析发现,灵芝CaM基因序列编码149个氨基酸,与其他物种CaM蛋白高度保守。该蛋白含有4个完整的EF-hand结构域,并且在每个EF-hand结构域中,都含有一个保守的D-x-D基序。进一步构建筛选了灵芝CaM沉默转化子,检测CaM在灵芝生长发育及次生代谢过程中的功能。结果显示,CaM沉默转化子中灵芝酸含量比WT降低约34%。沉默CaM后菌丝生长速率与WT相比降低40%。该结果说明CaM在灵芝生长及次生代谢过程中具有重要作用,为在真菌中研究CaM功能及调控途径提供参考。     

白藜芦醇对AngⅡ诱导的血管平滑肌细胞CaN的影响

目的：观察白藜芦醇（Res）对血管紧张素Ⅱ（AngⅡ）诱导的血管平滑肌细胞（VSMCs）增殖及细胞中钙调蛋白（CaM）和钙调神经磷酸酶（CaN）活性的影响,并探讨其机制。方法：体外培养兔主动脉VSMCs,用免疫细胞化学方法鉴定。建立AngⅡ诱导的VSMCs增殖模型。取生长良好的第4～8代VSMCs,随机分为对照组,AnsⅡ组（0．1μmol／L）,AngⅡ＋Res组（20,40,80,160）μmol／L。应用MTT法检测细胞增殖程度,考马斯亮兰法进行CaM定量,定磷法进行CaN活性测定。结果：成功培养兔VSMCs并传代,免疫细胞化学染色均呈阳性表达。AngⅡ组VSMCs的增殖程度、CaM和CaN活性较对照组增高（P〈0．05,P〈0．01）。AngⅡ＋Res组各组VSMCs的CaM和CaN活性较AngⅡ组显著下降（P〈0．01）。结论：在一定范围内,Res可降低AngⅡ诱导的VSMCs增殖程度,其机制可能与干预CaN依赖的信号转导途径有关。     

大麦类钙调磷酸酶B互作蛋白激酶基因克隆及表达分析

采用RT-PCR法从大麦抗旱品种‘甘啤7号’中克隆了1个类钙调磷酸酶B互作蛋白激酶(CIPK)基因HvCIPK1(GenBank登录号为JX679077)。序列分析表明,该基因全长1 359bp,编码的蛋白含有452个氨基酸,分子量为51.05kD,等电点为9.13。推断具有植物CIPK家族典型的功能结构域,在N端有一特异的催化结构域,该结构域在保守的氨基酸DFG和APE之间含有一个催化所需的活性环;同时C端区域存在一个独特的24个氨基酸组成的调节域,即NAF结构域。荧光定量分析结果表明,在PEG、NaCl和ABA处理下,HvCIPK1基因在大麦幼苗叶片中表达显著上调。推测HvCIPK1基因参与多种逆境信号的转导,可能在大麦的多种非生物胁迫响应中起着重要的作用。     

酿酒酵母细胞中钙离子信号传导途径的研究进展

酿酒酵母（SaccharomycescP增v括fdP）细胞可以通过ca2＋／钙调磷酸酶信号途径来应对许多外界环境胁迫。在交配信息素、盐或者其他环境压力存在的条件下,钙离子会通过细胞质膜上的未鉴定的钙转运蛋白x和M或者由Cchl和Midl组成的钙通道进入细胞质。胞质内钙离子浓度的增加会激活细胞质里的钙调磷酸酶（calcineurin）。钙调磷酸酶的一个非常重要的作用是去磷酸化细胞质内的转录因子Crzl,造成它快速地从细胞质转移到细胞核,从而诱导包括液泡膜上钙泵蛋白基因PMCl以及内质网膜和高尔基体膜上钙泵蛋白基因尸脚,在内的目标基因的表达。这两个钙泵蛋白和液泡膜上的Ca2＋／H＋交换蛋白Vcxl一起作用,将细胞质内的钙离子浓度控制在50～200nmol／L的正常生理浓度内．使细胞能够正常生长。该综述主要论述了酿酒酵母细胞内Ca2＋／钙调磷酸酶信号途径的最新研究进展。     

西伯利亚蓼钙调蛋白基因的克隆及其在盐胁迫下的表达

已从西伯利亚蓼叶中cDNA文库中获得的钙调蛋白EST序列,采用cDNA末端快速扩增（RACE）技术克隆了具有完整编码区的钙调蛋白基因的cDNA序列（GenBank登录号GQ988382）,命名为PsCaM。该基因全长615bp,编码区为450bp,编码149个氨基酸,5＇非翻译区为63bp,3＇非翻译区为102bp。同源性分析表明,该蛋白与其他植物钙调蛋白高度保守,氨基酸同源性高达98%。用实时荧光定量PCR研究3%NaHCO3胁迫下西伯利亚蓼基因表达的结果显示,自然条件下,该基因在叶中表达量最高,地下茎次之,茎中最低;盐胁迫下CaM在西伯利亚蓼的地下茎、茎和叶中均有表达,表达模式不同。     

盐桦BhNHX的克隆及其与CaM在胁迫下的协同表达

以盐桦组培苗为材料,通过RACE技术克隆了液泡膜Na /H 反向运输体基因BhNHX,采用DNAman软件和BLASTN对cDNA序列进行分析并与其他植物的NHX基因进行同源性比较,最后对BhNHX和钙调蛋白基因(CaM)在各种胁迫条件下的协同表达进行半定量的RT-PCR分析.结果显示:(1)获得了BhNHX的全长cDNA序列,包含1 623 bp的开放阅读框架,编码一个540个氨基酸的多肽,有11个推测跨膜区,与已报道的液泡膜Na /H 反向运输载体蛋白跨膜区数目一致;(2)BhNHX的核酸序列与其他植物NHX具有较高同源性,与葡萄NHX序列一致性达到76%;(3)BhNHX和钙调蛋白基因CaM可同时被盐、低温、干旱所诱导;但ABA只能较明显诱导BhNHX的表达,而对CaM的诱导表达不明显.研究表明,在盐桦受到盐、低温及干旱胁迫时BhNHX和CaM可能协同表达,而在ABA诱导时两者可能具有不同的表达调控模式.     

The secondary structure of calcineurin regulatory region and conformational change induced by calcium/calmodulin binding

The protein serine/threonine phosphatase calcineurin (CN) is activated by calmodulin (CaM) in response to intracellular calcium mobilization. A widely accepted model for CN activation involves displacement of the CN autoinhibitory peptide (CN(467-486)) from the active site upon binding of CaM. However, CN activation requires calcium binding both to the low affinity sites of CNB and to CaM, and previous studies did not dissect the individual contributions of CNB and CaM to displacement of the autoinhibitory peptide from the active site. In this work we have produced separate CN fragments corresponding to the CNA regulatory region (CNRR(381-521), residues 381-521), the CNA catalytic domain truncated at residue 341, and the CNA-CNB heterodimer with CNA truncated at residue 380 immediately after the CNB binding helix. We show that the separately expressed regulatory region retains its ability to inhibit CN phosphatase activity of the truncated CN341 and CN380 and that the inhibition can be reversed by calcium/CaM binding. Tryptophan fluorescence quenching measurements further indicate that the isolated regulatory region inhibits CN activity by occluding the catalytic site and that CaM binding exposes the catalytic site. The results provide new support for a model in which calcium binding to CNB enables CaM binding to the CNA regulatory region, and CaM binding then instructs an activating conformational change of the regulatory region that does not depend further on CNB. Moreover, the secondary structural content of the CNRR(381-521) was tentatively addressed by Fourier transform infrared spectroscopy. The results indicate that the secondary structure of CNRR(381-521) fragment is predominantly random coil, but with significant amount of beta-strand and alpha-helix structures.     

Preparation and characterization of a single-chain calcineurin-calmodulin complex

Calcineurin (CN), a Ca(2+)/calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca(2+)-regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM-CNB-CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB-CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn2+, Ni2+ and Mg2+. Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca2+.     

Function and structure of N-terminal and C-terminal domains of calcineurin B subunit

Calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, plays a critical role in T-cell activation by regulating the activity of NF-AT. CN is a heterodimer consisting of a catalytic subunit (CNA) and a Ca2+-binding regulatory subunit (CNB). CNB is composed of two global domains: the C-terminal domain (DC) and the N-terminal domain (DN), each containing two Ca2+ binding sites. In this study, using purified DN and DC derived from constructed expression systems, we revealed that intact CNB and DC can stimulate the phosphatase activity of CNA, about 2.2 and 1.6 times the phosphatase activity of CNA alone, respectively; DN itself has little effect on the phosphatase activity of CNA. Fluorescence spectroscopy of an ANS-hydrophobic fluorescence probe shows that binding of Ca2+ to CNB, DC or DN leads to exposure of the hydrophobic surface of the proteins and that the hydrophobicity of CNB is the greatest, that of DC is less, and that of DN is the least. The hydrophobic surface of CNB may be an important structural basis for stimulating CN phosphatase activity.     

The salt bridge of calcineurin is important for transferring the effect of CNB binding to CNA

Calcineurin (CN) is a heterodimer consisting of a catalytic subunit (CNA) and a regulatory subunit (CNB). The crystal structure shows that three residues or regions of CNA are mainly responsible for the interaction with CNB: the CNB binding helix (BBH), the N-terminus, and Glu53 that forms a salt bridge with Lys134 of CNB. In this report, we try to find the role that the salt bridge plays in the interaction between CNA and CNB. We found that mutation of Glu53 greatly reduced its responsiveness to CNB in the phosphatase assay and also that mutation of Lys134 of CNB affected its ability to activate the phosphatase activity of CNA. Structural analysis showed that disruption of the salt bridge affected the compact association of CNA and CNB. Thus, the salt bridge appears to help to stabilize CN and transfer the effects of CNB binding to CNA to activate its phosphatase activity.     

Effect of metal ions on the activity of the catalytic domain of calcineurin

Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI). It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions. Mn2+ and Ni2+ are the most crucial cation-activators for this enzyme. In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA alpha subunit and its catalytic domain (CNa) were cloned and expressed in E. coli. The effects of Mn2+, Ni2+ and Mg2+ on the catalytic activity of these purified proteins were examined. Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa. However, the activation degree of CNa by the metal ions was much higher than that of CNA. In term of different metal ions, the activating extents to CNA and CNa were different. To CNA, the activating order from high to low was Mg2+ > > Ni2+ > Mn2+, but Mn2+ > Ni2+ > > Mg2+ to CNa. No effect of CaM/Ca2+ and CNB/Ca2+ on the activity of CNa was observed in our experiments. Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified. These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa. The activating extents of metal ions via catalytic domain were higher than that via regulating fragment. The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn2+, Ni2+ and Mg2+.     

TRAF3 negatively regulates calcineurin-NFAT pathway by targeting calcineurin B subunit for degradation

Calcineurin (CN) is the only serine/threonine specific protein phosphatase regulated by Ca(2+) /calmodulin (CaM), which is composed of catalytic A subunit (CNA) and regulatory B subunit (CNB). Tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3) is an essential component in the Toll like receptors and TNF receptors (TNFRs) pathways. The TRAF domain of TRAF3 interacts with a large range of proteins, which share consensus sequences known as TRAF interacting motifs (TIMs). By sequence alignment, we identified two potential TIMs in CNB. However, the relation between TRAF3 and CN has not been reported before. To explore this, we highly expressed the former insoluble TRAF domain of TRAF3 in soluble form by using CaM fusion system for the first time. We demonstrated that the TRAF domain of TRAF3 interacted with CNB. On further investigation, over-expression of TRAF3 inhibited endogenous CN's activity, which decreased NFAT reporter activity and IL-2 production. Knock-down of TRAF3 partially enhanced CN's activity. The possible mechanism was that TRAF3 functioned as ubiquitin E3 ligase for CN and promoted its degradation. ? 2012 IUBMB IUBMB Life IUBMB Life, 64(9): 748-756, 2012.     

A renewed model of CNA regulation involving its C-terminal regulatory domain and CaM

Calcineurin is composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). CNA contains the catalytic domain and three regulatory domains: a CNB-binding domain (BBH), a C-terminal calmodulin-binding domain (CBD), and an autoinhibitory domain (AID). We constructed a series of mutants of CNA to explore the regulatory role of its C-terminal regulatory domain and CaM. We demonstrated a more precise mechanism of CNA regulation by C-terminal residues 389-511 in the presence of CNB. First, we showed that residues 389-413, which were identified in previous work as constituting a CaM binding domain (CBD), also have an autoinhibiting function. We also found that residues 389-413 were not sufficient for CaM binding and that the CBD comprises at least residues 389-456. In conclusion, two distinct segments of the C-terminal regulatory region (389-511) of CNA inhibit enzyme activity: residues 389-413 interact with the CNB binding helix (BBH), and residues 457-482 with the active center of CNA.     

Identification of the site on calcineurin phosphorylated by Ca2+/CaM-dependent kinase II: modification of the CaM-binding domain

The catalytic subunit of the Ca2+/calmodulin- (CaM) dependent phosphoprotein phosphatase calcineurin (CN) was phosphorylated by an activated form of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) incorporating approximately 1 mol of phosphoryl group/mol of catalytic subunit, in agreement with a value previously reported (Hashimoto et al., 1988). Cyanogen bromide cleavage of radiolabeled CN followed by peptide fractionation using reverse-phase high-performance liquid chromatography yielded a single labeled peptide that contained a phosphoserine residue. Microsequencing of the peptide allowed both the determination of the cleavage cycle that released [32P]phosphoserine and the identity of amino acids adjacent to it. Comparison of this sequence with the sequences of methionyl peptides deduced from the cDNA structure of CN (Kincaid et al., 1988) allowed the phosphorylated serine to be uniquely identified. Interestingly, the phosphoserine exists in the sequence Met-Ala-Arg-Val-Phe-Ser(P)-Val-Leu-Arg-Glu, part of which lies within the putative CaM-binding site. The phosphorylated serine residue was resistant to autocatalytic dephosphorylation, yet the slow rate of hydrolysis could be powerfully stimulated by effectors of CN phosphatase activity. The mechanism of dephosphorylation may be intramolecular since the initial rate was the same at phosphoCN concentrations of 2.5-250 nM.     

Kaempferol: a new immunosuppressant of calcineurin

Calcineurin (CN), the Ca(2+)/calmodulin (CaM)-dependant protein phosphatase, is the target for immunosuppressive drugs cyclosporine A (CsA) and FK506. These immunosuppressants can inhibit CN activity after binding with respective immunophilins. Based on the model of screening by using p-nitrophenyl phosphate as a substrate for preliminary screening and (32)P-labeled 19-residue phosphopeptide as a specific substrate for final determination, we found Kaempferol, a natural flavonol, could inhibit CN activity in purified enzyme and Jurkat T-cells. Unlike CsA and FK506, CN inhibition by kaempferol is independent of matchmaker protein and the inhibitory manner is noncompetitive. Through investigation of inhibitions for CNA and a series of its truncated mutants, we suggested that Kaempferol could directly act on the catalytic domain. Data also indicated that the CN inhibition by kaempferol could be enhanced when the enzyme was activated in the presence of CaM and CNB. CNB is necessary for mediating inhibition of enzyme by kaempferol. The result of RT-PCR also indicated that kaempferol had an inhibitory activity against IL-2 gene expression in activated Jurkat cells. All data suggested that kaempferol could be a new immunosuppressant of CN.     

The complex structure of calmodulin bound to a calcineurin peptide

The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25-residue peptide taken from the CaMBD, joined by a 5-glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25-residue CaMBD peptide without the glycine linker at a resolution of 2.1 A. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N-lobe from one molecule and C-lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully-designed CaM-sepharose pull-down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg-408, Val-409, and Phe-410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N- and C-lobes, ultimately keeping these lobes apart and forcing "head-to-tail" dimerization to attain the requisite N- and C-lobe pairing for CaMBD binding.