Calpain对细胞骨架蛋白tau降解作用的研究(英)

Calpain是钙依赖性中性蛋白酶,根据其对钙敏感性的不同,可分为m-和μ-calpain两型.分别用不同浓度CaCl₂溶液孵育Wistar大鼠脑皮质匀浆液,并用蛋白质印迹和定量图像分析技术检测不同亚型calpain对tau蛋白的降解作用.研究发现：在37℃用1 mmol/L Ca²⁺孵育底物15 min,可见tau蛋白明显降解,并在分子质量为29 ku处出现tau蛋白降解片段;当Ca²⁺浓度为5 mmol/L时,tau蛋白几乎全部被降解;这种tau蛋白降解可被calpain特异性抑制剂完全逆转.进一步的研究发现,分别用μ-calpain抑制剂（0.05 μmol/L calpastatin）,m-calpain抑制剂（100 μmol/L calpain inhibitor Ⅳ）或总calpain抑制剂（552 μmol/L calpeptin）与1 mmol/L Ca²⁺共同孵育Wistar大鼠脑皮质匀浆液,Ca²⁺激活的tau蛋白降解分别被抑制8.6%,92.5%和97.8%.结果表明一定浓度的Ca²⁺可同时激活μ-calpain和m-calpain,这两种亚型calpain均参与降解tau蛋白,但m-calpain的作用比μ-calpain更强.     

1型糖尿病葡萄糖代谢与胰岛素信号传导途径异常导致大鼠海马Alzheimer样tau蛋白过度磷酸化修饰

Tau蛋白过度磷酸化是Alzheimer病(Alzheimer disease, AD)的一个重要病理特征.采用 I 型糖尿病大鼠模型,研究胰岛素信号传导途径及葡萄糖代谢失调对tau蛋白过度磷酸化的形成机制进行探讨.以同龄Wistar大鼠做对照（CTL）,胰腺大部分切除造低胰岛素组（PX）,STZ较大剂量一次性注射造1型糖尿病模型即低胰岛素高血糖组（T1DM）.葡萄糖氧化酶法检测血浆血糖,放免法检测血浆胰岛素,蛋白质印迹分析海马内总tau蛋白及tau蛋白上部分位点（Ser199、Thr212、Ser214、Ser396及Ser422）的磷酸化及神经细胞膜上葡萄糖转运子3（Glucose transport 3,GLUT3）水平.γ-32P-ATP和特异性底物肽检测海马内胰岛素信号传导系统中的关键酶糖原合成酶激酶-3β（Glycogen synthase kinase-3β, GSK-3β）活性.发现3组大鼠海马回总tau蛋白水平无显著差异,但以高血糖、低胰岛素血症为特征的T1DM组在tau蛋白Ser199、Thr212、Ser214、Ser396及Ser422位点上,呈现过度磷酸化状态,以低胰岛素血症为特征而血糖正常的PX组在位点Ser199、Thr212及Ser396上磷酸化程度比CTL组显著上升, 在位点Ser214及 Ser422上的磷酸化程度的改变不显著;T1DM及PX组大鼠海马 GSK-3β活性显著高于CTL组, 而GLUT3水平在T1DM和PX组均降低, 尤以T1DM组降低更显著.研究结果显示,胰岛素水平低下可能通过激活GSK-3β和下调细胞内葡萄糖代谢的双重作用引起脑内tau蛋白过度磷酸化.     

cdk-5过度表达对大鼠tau蛋白磷酸化及空间记忆的影响

Alzheimer病(AD)中, 异常过度磷酸化的tau蛋白会导致细胞骨架的异常并与神经元的死亡有关. 在体外, 细胞周期蛋白依赖性蛋白激酶5(cdk-5)能在大多数AD相关位点磷酸化tau蛋白. 旨在整体水平研究cdk-5过度表达对大鼠微管相关蛋白 tau的磷酸化及空间记忆的影响. 结果显示, 在大鼠海马区转染cdk-5基因, 24 h后其局部表达增加, 并使得抗体tau-1显色减弱, PHF-1和12e8显色增强, 提示tau蛋白在Ser199/202, Ser396/404和Ser262/356位点过度磷酸化. 此外, 在水迷宫测试中, cdk-5转染鼠寻找安全平台所需时间比对照鼠明显延长, 而转染后48 h cdk-5的表达较24 h时下降, 同时伴有tau蛋白磷酸化程度的下降和空间记忆能力的改善. 这些结果提示整体水平的cdk-5的过度表达会导致大鼠的空间记忆损伤, 而过度磷酸化的tau蛋白可能参与了该病理过程.     

微管相关蛋白tau在动物死亡后被快速去磷酸化

tau蛋白是神经细胞中主要的微管相关蛋白, 它的异常过度磷酸化被认为是阿尔茨海默病 (AD) 致病过程中的关键因素. 由于法律、社会、家庭等诸多因素使得获取的人脑组织标本常常在死亡后2~3 h以上,因此了解死亡不同时间后tau蛋白磷酸化的改变,对研究tau蛋白的功能及在AD致病过程中作用显得十分重要. 用位点特异的、磷酸化依赖的抗tau蛋白抗体检测正常大鼠脑中tau蛋白磷酸化程度及死亡后其磷酸化的变化情况,再用非同位素的点印迹技术测定鼠脑中tau蛋白激酶、磷酸酶在不同温度下的活性. 结果发现,正常鼠脑中tau蛋白除了Ser262,Ser409,Ser422外,在Thr181,Ser199,Ser202,Thr205,Thr212,Ser214,Thr217,Ser396和Ser404存在不同程度的磷酸化,并且在死亡后3 h,出现tau的多位点的去磷酸化及tau蛋白迁移加快,6 h后更为明显,但tau蛋白水平即使在大鼠死亡后6 h,仍未见有明显的改变. 用点印迹测定蛋白激酶和磷酸酶活性结果显示,tau蛋白激酶、磷酸酶活性均有温度依赖性降低,在25℃时激酶活性降低远大于磷酸酶活性的降低,tau蛋白在死亡后的快速去磷酸化与相对高的磷酸酶作用有关.     

C-激酶在跨膜信息传递中的作用

C-激酶(PKC)在跨膜信息传递中具有重要作用,其作用过程与A-激酶、G-激酶、Ca²⁺·CaM蛋白激酶相互区别而又紧密联系。C-激酶系通过催化多种蛋白特别是胰岛素受体,生长因子受体、钙调蛋白等生理活性蛋白的Ser/Thr残基磷酸化,以调节细胞代谢,分化、生长、增殖乃至癌变。     

Ⅱ型糖尿病大鼠海马Alzheimer病样Tau蛋白过度磷酸化修饰及机制探讨

Tau蛋白过度磷酸化是Alzheimer病 (Alzheimer′s disease, AD) 的一个重要特征.本研究检测了Ⅱ型糖尿病大鼠海马tau蛋白磷酸化水平,对其形成机制进行探讨. 以同龄正常Wistar大鼠作为对照,高脂高蛋白高糖饮食加小剂量链脲佐菌素（streptozotocin,STZ）注射诱导造Ⅱ型糖尿病模型（T2DM组）.放免法检测血浆胰岛素;葡萄糖氧化酶法检测血浆葡萄糖;蛋白质印迹技术检测各组大鼠海马内总tau蛋白、tau蛋白上部分位点磷酸化、神经细胞膜上胰岛素受体及葡萄糖转运子3（glucose transport 3,GLUT3）水平;表面等离子共振技术（surface plasmon resonance, SPR）检测细胞膜上胰岛素受体与血浆胰岛素结合力;γ32-P标记的ATP和特异性底物肽检测海马内胰岛素信号传导系统中的关键酶糖原合酶激酶-3β（glycogen synthase kinase-3β, GSK-3β）活性.结果显示,T2DM组血浆血糖、血浆胰岛素及运用HOMA-IR公式计算的胰岛素抵抗指数显著高于对照组.蛋白质印迹结果显示两组大鼠海马回总tau蛋白水平无差异;T2DM组中tau蛋白在Ser199、Thr212、Ser214、Thr217、Ser396及Ser422位点上的磷酸化水平均显著高于对照组;T2DM组海马神经细胞膜上胰岛素受体水平及与胰岛素结合的功能均显著低于对照组;GSK-3β活性检测结果显示,T2DM组大鼠模型海马回中GSK-3β活性明显增高.研究结果表明,Ⅱ型糖尿病中由于胰岛素抵抗导致GSK-3β激活从而出现AD样tau蛋白的过度磷酸化,葡萄糖代谢紊乱也可能在tau蛋白的过度磷酸化起一定作用.     

p16INK4a基因的功能及其调控

p16^INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16^INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16^INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16^INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

钙信号途径参与小斑病菌致病过程的调控*

为确定Ca²⁺信号途径与玉米小斑病菌致病过程的相关性,用可从不同位点阻断Ca²⁺信号转导途径的抑制剂分别处理小斑病菌的分生孢子,结果表明:Ca²⁺螯合剂EGTA、Ca²⁺通道抑制剂Verapam il、影响钙调素与钙调素依赖蛋白激酶作用位点的抑制剂KN-93,随着浓度的增加,对孢子萌发和附着胞形成过程的抑制作用明显增强;同一浓度下,抑制剂对附着胞形成过程的抑制作用大于孢子萌发过程;抑制剂可使附着胞形态明显变小甚至不能形     

褪黑素对异丙肾上腺素诱导大鼠tau蛋白过度磷酸化的预防作用

异常过度磷酸化的微管相关蛋白tau是阿尔茨海默病(Alzheimer's disease,AD)患者大脑中神经原纤维缠结的主要组成部分.迄今为止,尚无有效的措施阻止tau蛋白的过度磷酸化.为探讨褪黑素(melatonin,Mel)对AD样tau蛋白过度磷酸化的预防作用,我们以β受体激动剂异丙肾上腺素(isoproterenol,IP)来复制AD样tau蛋白过度磷酸化的动物模型,在大鼠双侧海马注射IP前,以褪黑素作为保护组药物,于腹腔连续注射5 d.应用磷酸化位点特异性抗体(PHF-1和Tau-1)作免疫印迹和免疫组织化学检测tau蛋白的磷酸化水平,并用非磷酸化依赖的总tau蛋白抗体(111e)进行标准化.免疫印迹结果显示在注射IP 48 h后,tau蛋白在PHF-1表位的免疫反应显著增强,在Tau-1表位显著减弱,表明tau蛋白在Ser396/Ser404(PHF-1)和Ser199/Ser202(Tau-1)位点有过度磷酸化.免疫组织化学染色结果与免疫印迹结果相似,主要检测到在大鼠海马CA3区的神经纤维有tau蛋白过度磷酸化.褪黑素预处理大鼠可有效地阻止IP诱导tau蛋白在Tau-1和PHF-1位点的过度磷酸化.上述结果提示褪黑素可预防大鼠脑组织中由异丙肾上腺素引起的AD样tau蛋白的过度磷酸化.     

35S标记重组植物钙调素的制备方法

利用³⁵S标记的氨基酸混合物喂养工程菌,成功地制备了³⁵S标记的拟南芥钙调素亚型2（³⁵S-ACaM2）,对其纯度、放射活度、电泳行为及其灵敏性等进行了检测.结果表明从工程菌中制备的³⁵S-ACaM2纯度高、放射活度高、Ca²⁺与EGTA存在时的电泳行为与未标记的ACaM2相同,可作为一种高灵敏性的探针用于检测钙调素结合蛋白.     

Isolation and characterization of biliprotein aggregates from Acaryochloris marina, a Prochloron-like prokaryote containing mainly chlorophyll d

Phycobiliprotein aggregates were isolated from the prokaryote Acaryochloris marina, containing chlorophyll d as major pigment. In the electron microscope the biliprotein aggregates appear as rod-shaped structures of 26.0×11.3 nm, composed of four ring-shaped subunits 5.8 nm thick and 11.7 nm in diameter. Spectral data indicate that the aggregates contain two types of biliproteins: phycocyanin and an allophycocyanin-type pigment, with very efficient energy transfer from the phycocyanin- to allophycocyanin-type constituent. The chromophore-binding polypeptides of the pigments have apparent molecular masses of 16.2 and 17.4 kDa. They crossreact with antibodies against phycocyanin and allophycocyanin from a red alga.     

Polypeptide sequence of the chlorophyll a/b/c-binding protein of the prasinophycean alga Mantoniella squamata

The primary structure of the Chla/b/c-binding protein from Mantoniella squamata is determined. This is the first report that protein sequencing reveals one modified amino acid resulting in a LHCP-specific TFA-cleavage site. The comparison of the sequence of Mantoniella with other Chla/b-and Chla/c-binding proteins shows that the modified amino acid is located in a region which is highly conserved in all these proteins. The alignment also reveals that the LHCP of Mantoniella is related to the Chla/b-binding proteins. Finally, possible Chl-binding regions are discussed.Abbreviations a.m.u. atomic mass unit - LHC light-harvesting complex - LHC II major LHC of Photosystem II - LHCP light-harvesting chlorophyll-binding protein - LSIMS liquid secondary ion mass spectrometry - TFA trifluoroacetic acid     

Phosphorylation of P400 Protein by Cyclic AMP-Dependent Protein Kinase and Ca2+/Calmodulin-Dependent Protein Kinase II

Purified P400 protein was phosphorylated by both purified Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Because P400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P400 protein using crude mitochondrial and microsomal fractions (P2/P3 fraction). Incubation of the P2/P3 fraction from mouse cerebellum with cyclic AMP or the catalytic subunit of A-kinase stimulated the phosphorylation of P400 protein. The phosphorylation of P400 protein was not observed in the P2/P3 fraction from mouse forebrain. Cyclic AMP and A-kinase enhanced the phosphorylation of several proteins, including P400 protein, suggesting that P400 protein is one of the best substrates for A-kinase in the P2/P3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P2/P3 fraction, the phosphorylation of P400 protein was weak. Immunoprecipitation with the monoclonal antibody to P400 protein confirmed that the P400 protein itself was definitely phosphorylated by the catalytic subunit of A-kinase and CaM kinase II. A-kinase phosphorylated only the seryl residue in P400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P400 protein, which migrated at the same position on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that in the P2/P3 fraction. Incubation of the cultured cerebellar cells with [32P]orthophosphate resulted in the labeling of P400 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of Purified Rat Striatal Tyrosine Hydroxylase by Ca2+/Calmodulin-Dependent Protein Kinase II: Effect of an Activator Protein

The phosphorylation of tyrosine hydroxylase, purified from rat striatum, was investigated using purified Ca2+/calmodulin (CaM)-dependent protein kinase II. This kinase catalyzed the Ca2+-dependent incorporation of up to 0.8 mol 32PO4/mol tyrosine hydroxylase subunit (62 kilodaltons). Reverse-phase high-performance liquid chromatography mapping of tryptic 32P-peptides established that the Ca2+/CaM-dependent protein kinase II phosphorylated a different serine residue than was phosphorylated by the cyclic AMP-dependent protein kinase. Limited proteolysis sequentially reduced the subunit Mr from 62 to 59 kilodaltons and finally to 57 kilodaltons, resulting in loss of the site phosphorylated by the Ca2+/CaM-dependent protein kinase II, but not the site phosphorylated by the cyclic AMP-dependent protein kinase. Phosphorylation by the Ca2+/CaM-dependent protein kinase II had little direct effect on the kinetic properties of tyrosine hydroxylase, but did convert it to a form that could be activated twofold by addition of an activator protein. This heat-labile activator protein increased the Vmax without affecting the Km for the pterin cofactor. This effect was specific in that the activator protein was without effect on nonphosphorylated tyrosine hydroxylase or on tyrosine hydroxylase phosphorylated by the cyclic AMP-dependent protein kinase. These results are consistent with the hypothesis that the "Vmax-type" activation of tyrosine hydroxylase observed upon depolarization of neural and adrenal tissues may be mediated by the Ca2+/CaM-dependent protein kinase II.     

RGS7 Attenuates Signal Transduction Through the Gαq Family of Heterotrimeric G Proteins in Mammalian Cells

Abstract: The RGS proteins are a recently discovered family of G protein regulators that have been shown to act as GTPase-activating proteins (GAPs) on the G_αi and G_αq subfamilies of the heterotrimeric G proteins. Here, we demonstrate that RGS7 is a potent GAP in vitro on G_αi1 and G_αo heterotrimeric proteins and that RGS7 acts to down-regulate G_αq-mediated calcium mobilization in a whole-cell assay system using a transient expression protocol. This RGS protein and RGS4 are reported to be expressed predominantly in brain, and in situ hybridization studies have revealed similarities in the regional distribution of RGS and G_αq mRNA expression. Our findings provide further evidence to support a functional role for RGS4 and RGS7 in G_αq-mediated signaling in the CNS.     

Characterization of PhPRP1, a histidine domain arabinogalactan protein from Petunia hybrida pistils

An arabinogalactan protein, PhPRP1, was purified from Petunia hybrida pistils and shown to be orthologous to TTS-1 and TTS-2 from Nicotiana tabacum and NaTTS from Nicotiana alata. Sequence comparisons among these proteins, and CaPRP1 from Capsicum annuum, reveal a conserved histidine-rich domain and two hypervariable domains. Immunoblots show that TTS-1 and PhPRP1 are also expressed in vegetative tissues of tobacco and petunia respectively. In contrast to the molecular mass heterogeneity displayed by the pistil proteins, the different isoforms found in seedlings, roots, and leaves each has a discrete size (37, 80, 160, and 200 kDa) on SDS-PAGE gels. On the basis of their chemistry, distinctive domain architecture, and the unique pattern of expression, we have named this group of proteins HD-AGPs (histidine domain-arabinogalactan proteins).     

Uncoupling of Rat Cerebral Cortical α2-Adrenoceptors from GTP-Binding Proteins by N-Ethylmaleimide

Pretreatment of membranes from rat cerebral cortex with N-ethylmaleimide (NEM) decreased [3H]-clonidine binding in a concentration-dependent manner. The Bmax values of high-affinity sites for [3H]clonidine were reduced by 50 microM NEM treatment. Treatment with 500 microM NEM diminished the sum of Bmax of both high- and low-affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM-treated membranes. The addition of purified GTP-binding proteins caused an increase in the binding to the membranes pretreated with 50 microM NEM, but did not increase [3H]-clonidine binding in membranes treated with 500 microM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)-catalyzed ADP ribosylation of membrane-bound (41,000-dalton) and purified (39,000/41,000-dalton) GTP-binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, alpha 2-adrenoceptor, and GTP-binding protein. One is a highly sensitive site to NEM (a concentration range of 1-50 microM), which is probably a cysteine residue, IAP-catalyzed ADP-ribosylating site on the alpha-subunit of GTP-binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1-1 mM), and are the binding domain of agonist and/or the coupling domain of GTP-binding protein on the alpha 2-adrenoceptor. In addition, Ki-ras p21 protein may lack the capacity to couple with the alpha 2-adrenoceptor.     

Characterization of proteins from arthrodial membranes of the lobster, Homarus americanus

A total of six proteins from the abdominal arthrodial membrane (intersegmental membrane) of the lobster, Homarus americanus, were purified and their amino acid sequences were determined by a combination of mass spectrometry and Edman degradation. The proteins are acidic with pI-values close to 4 and they all have molecular masses ≈12 kDa. The sequences of five of the proteins differ in only a few residues, while the sixth protein differs from the others in more than half of the positions. Only little similarity is observed between the sequences of the arthrodial membrane proteins and those of proteins purified from the calcified parts of the exoskeleton of H. americanus. The arthrodial membrane proteins contain the Rebers-Riddiford consensus sequence common in proteins from insect cuticles. Comparison of the complete sequences to the sequences available in databases shows that the lobster membrane proteins are more closely related to proteins from insect pliant cuticles than to proteins derived from cuticles destined for sclerotization. Characteristic features in the protein sequences are discussed, and it is suggested that the various sequence regions have specific roles in determining the mechanical properties of arthrodial membranes.