Caspase-3 对磷酸化 tau 蛋白截断作用的研究

磷酸化 tau 是阿尔茨海默病 (Alzheimer's disease , AD) 的特征性病理改变———神经原纤维缠结 (neurofibrillary tangles , NFTs) 的主要组成部分 . 最近的研究显示： NFT 存在 Glu391 和 Asp421 位点被截断的 tau 片段,然而, tau 蛋白的磷酸化是否会影响 caspase-3 的切割作用尚不清楚 . 首先纯化重组 tau 蛋白,然后利用蛋白激酶 A (PKA) 、钙 / 钙调蛋白依赖性蛋白激酶Ⅱ (CaMK Ⅱ ) 和乳鼠海马组织抽提液对其磷酸化,并用 caspase-3 对不同磷酸化的 tau 蛋白进行切割,比较 caspase-3 对非磷酸化和不同蛋白激酶磷酸化的 tau 蛋白的切割特性 . 结果显示：除切割非磷酸化 tau 蛋白外, caspase-3 在体外可分别切割被 PKA 、 CaMK Ⅱ和乳鼠海马组织抽提液磷酸化的 tau 蛋白 . 这一结果提示：磷酸化修饰的 tau 蛋白仍然是 caspase-3 的底物 .     

饥饿对小鼠脑中tau蛋白磷酸化和O-GlcNAc糖基化的影响

为了探讨大脑中葡萄糖摄取和代谢障碍在阿尔茨海默病(Alzheimer$sdisease,AD)神经退行性病变中的作用,将昆明种小鼠进行饥饿和再喂食处理,并使用多种磷酸化tau蛋白特异性的抗体和蛋白O-GlcNAc糖基化特异性抗体进行检测,观察饥饿及恢复喂养后不同时间点大脑皮质中tau蛋白糖基化及多个位点磷酸化的变化.结果显示:饥饿处理引起小鼠大脑皮质中总蛋白和tau蛋白的O-GlcNAc糖基化水平降低,同时tau蛋白磷酸化水平升高,饥饿引起的tauO-GlcNAc糖基化和磷酸化改变均在恢复进食后逆转成正常水平.该研究结果提示:大脑中tau蛋白的磷酸化和O-GlcNAc糖基化之间存在相互调节,脑中葡萄糖代谢障碍可能通过下调tau蛋白O-GlcNAc糖基化水平使tau蛋白产生异常过度磷酸化,进而促发AD的病理进程.这一结果为在早期阶段通过逆转tau蛋白异常过度磷酸化治疗AD成为可能提供了实验基础.     

tau蛋白异常与阿尔茨海默病的关系

阿尔茨海默病(Alzheimer disease,AD)是最常见的一种老年性痴呆症,以进行性记忆丧失和认知功能障碍为临床特征,神经原纤维缠结(neurofibrillary tangle,NFT)为其主要病理学特征之一,tau蛋白的各种异常与NFT的形成密切相关。对tau蛋白的各种异常导致AD发生的研究已取得重要进展,包括tau蛋白的异常磷酸化、异常糖基化、异常截断作用及基因突变等。本文旨在概述tau蛋白的各种异常改变及其可能机制。     

蛋白质O-GlcNAc糖基化修饰对tau蛋白磷酸化修饰的影响

蛋白质的O位N-乙酰葡萄糖胺(O-GlcNAc)糖基化修饰是一种新近发现的广泛存在于细胞核蛋白与细胞浆蛋白的蛋白质翻译后修饰.其性质与经典的膜蛋白和分泌蛋白的糖基化修饰不同,而与蛋白质磷酸化修饰更相似.O-GlcNAc糖基化和磷酸化均修饰tau蛋白的丝氨酸和苏氨酸残基,通过改变O-GlcNAc糖基化供体底物浓度以及其关键酶活性等方法,改变分化后成神经细胞样的PC12细胞中的蛋白质O-GlcNAc糖基化修饰水平,然后用特异性识别不同位点磷酸化的tau蛋白抗体,进行蛋白质印迹分析来检测tau蛋白磷酸化水平的变化.结果发现细胞内蛋白质O-GlcNAc糖基化对tau蛋白磷酸化的影响,在不同的磷酸化位点其影响不同.增加蛋白质O-GlcNAc糖基化修饰导致tau蛋白大多数磷酸位点的磷酸化水平降低,反之亦然.这些结果说明,tau磷酸化在大多数位点受到O-GlcNAc糖基化修饰的负性调节.这一研究为阐明调节tau蛋白磷酸化水平的机理和阿尔茨海默病脑中tau异常过度磷酸化的分子机制提供了新的线索.     

阿尔兹海默病中tau蛋白磷酸化调节

细胞内高度磷酸化tau蛋白形成的神经纤维缠结是阿尔兹海默病的主要病理特征之一。过度磷酸化的tau蛋白将引起细胞内微管的紊乱,从而造成神经元突触连接的丢失。Tau蛋白的磷酸化受到多种因素的影响,这些因素的失常将会导致tau蛋白的异常磷酸化。Tau蛋白的基本功能和结构、翻译后的主要修饰以及蛋白激酶和磷酸酯酶的调节,在阿尔兹海默病理以及预防治疗中发挥重要作用。     

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.     

Tau蛋白在阿尔茨海默病中的作用

阿尔茨海默病(AD)是非常普遍的神经变性性疾病并且是老年人痴呆的主要原因。AD患者的症状特点包括进行性的认知障碍、记忆丧失和行为障碍,与大脑中的病理变化密切相关。AD现成为全球最严重的健康和社会经济问题。在AD患者脑中神经纤维网或神经营养障碍的过程中存在tau蛋白的异常。tau蛋白丧失其促微管组装的生物学功能,导致细胞骨架的破坏、丝状物形成和神经缠结,轴突运输损害,进而导致突触蛋白失去功能和神经退行性病变。其数量和结构的改变将会影响其功能而且会出现异常聚集。调节Tau蛋白的异常聚集的分子机制主要是一些翻译后修饰使其结构及构象发生变化。因此,异常磷酸化和截断的tau蛋白作为tau蛋白病理过程的关键机制而引起学者关注。本文描述了tau蛋白的结构和功能及其在AD中的主要病理变化,同时在本文中还涉及到磷酸化的tau蛋白是神经元对氧化应激的代偿反应这一观点。对tau蛋白进行更加全面的解读。     

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.     

丝氨酸/苏氨酸蛋白磷酸酯酶在tau蛋白异常磷酸化中的作用

Tau蛋白过度磷酸化在阿尔采末病（Alzheimer’s disease,AD）发病过程中发挥重要作用,抑制蛋白磷酸酯酶活性,可诱导tau的过度磷酸化和聚积。本文拟就近年来蛋白磷酸酯酶在tau蛋白异常磷酸化中的作用作一综述。     

钙调磷酸酶对阿尔茨海默病中tau蛋白异常磷酸化的作用

100年前,阿尔茨海默病（Alzheimer disease,AD）被首次报道,它是一种以进行性认知障碍和记忆力损伤为主要临床特征的中枢神经系统退行性疾病,是最常见的一种老年性痴呆.由异常磷酸化的tau蛋白构成的神经元纤维缠结（neurofibrillary tangle,NFT）是AD最重要的病理学改变之一.钙调磷酸酶（calcineurin,CN）是脑组织中含量最高的一种丝氨酸/苏氨酸蛋白磷酸酶,其活性依赖于Ca2＋和钙调蛋白.最近的研究显示,CN在脑中与NFT共定位,CN活性的下降能够导致tau蛋白在多个AD特征性位点的异常磷酸化,进而形成NFT.因而,CN活性的缺陷与AD的发生密切相关.CN活性缺陷导致的tau蛋白异常磷酸化具有可逆性,CN激活剂有望在其中扮演重要的角色.     

阿尔次海默病tau蛋白异常修饰与其功能的关系

阿尔次海默病易溶型胞浆tau和难溶型双螺旋丝中的tau均被异常磷酸化和异常糖基化修饰.异常修饰的tau丧失其促微管组装活性,用不同蛋白磷酸酯酶对难溶型双螺旋丝中的tau去磷酸化处理后可不同程度恢复其促微管组装生物学活性.单纯去糖基化处理只在很小限度恢复tau的功能,但去糖基化预处理可增强去磷酸化对tau上述活性的恢复.提示:a.tau的异常磷酸化是导致其功能活性丧失的直接因素,而糖基化修饰可能通过对其结构的影响而间接对tau功能活性发挥作用;b.蛋白磷酸酯酶可部分抑制和逆转阿尔次海默病的脑病理损伤.     

Brain Levels of Microtubule-Associated Protein τ Are Elevated in Alzheimer''s Disease: A Radioimmuno-Slot-Blot Assay for Nanograms of the Protein

The microtubule-associated protein tau, which stimulates the assembly of alpha-beta tubulin heterodimers into microtubules, is abnormally phosphorylated in Alzheimer's disease (AD) brain and is the major component of paired helical filaments. In the present study, the levels of tau and abnormally phosphorylated tau were determined in brain homogenates of AD and age-matched control cases. A radioimmuno-slot-blot assay was developed, using a primary monoclonal antibody, Tau-1, and a secondary antibody, antimouse 125I-immunoglobulin G. To assay the abnormally phosphorylated tau, the blots were treated with alkaline phosphatase before immunolabeling. The levels of total tau were about eightfold higher in AD (7.3 +/- 2.7 ng/micrograms of protein) than in control cases (0.9 +/- 0.2 ng/micrograms), and this increase was in the form of the abnormally phosphorylated protein. These studies indicate that the abnormal phosphorylation--not a decrease in the level of tau--is a likely cause of neurofibrillary degeneration in AD.     

Alzheimer neurofibrillary degeneration: significance, etiopathogenesis, therapeutics and prevention

Alzheimer disease (AD) is multi-factorial and heterogeneous. Independent of the aetiology, this disease is characterized clinically by chronic and progressive dementia and histopathologically by neurofibrillary degeneration of abnormally hyperphosphorylated tau seen as intraneuronal neurofibrillary tangles, neuropil threads and dystrophic neurites, and by neuritic (senile) plaques of beta-amyloid. The neurofibrillary degeneration is apparently required for the clinical expression of AD, and in related tauopathies it leads to dementia in the absence of amyloid plaques. While normal tau promotes assembly and stabilizes microtubules, the abnormally hyperphosphorylated tau sequesters normal tau, MAP1 and MAP2, and disrupts microtubules. The abnormal hyperphosphorylation of tau also promotes its self-assembly into tangles of paired helical and or straight filaments. Tau is phosphorylated by a number of protein kinases. Glycogen synthase kinase-3 (GSK-3) and cyclin dependent protein kinase 5 (cdk5) are among the kinases most implicated in the abnormal hyperphosphorylation of tau. Among the phosphatases which regulate the phosphorylation of tau, protein phosphatase-2A (PP-2A), the activity of which is down-regulated in AD brain, is by far the major enzyme. The inhibition of abnormal hyperphosphorylation of tau is one of the most promising therapeutic targets for the development of disease modifying drugs. A great advantage of inhibiting neurofibrillary degeneration is that it can be monitored by evaluating the levels of total tau and tau phosphorylated at various known abnormally hyperphosphorylated sites in the cerebrospinal fluid of patients, obtained by lumbar puncture. There are at least five subgroups of AD, each is probably caused by a different etiopathogenic mechanism. The AD subgroup identification of patients can help increase the success of clinical trials and the development of specific and potent disease modifying drugs.     

肝素对人类神经tau蛋白分子聚集及磷酸化的影响

在老年性痴呆患者的脑中,肝素与超磷酸化的tau蛋白共存[7].采用NCLK(neuronalcdc2-likekinase)及PP2B(phosphoproteinphosphatase2B)在含肝素的体系中对人类神经tau蛋白进行磷酸化和脱磷酸化,结果表明,肝素具有促进tau蛋白被磷酸化的功能,并促进该蛋白磷酸化分子二聚体的形成和单体的减少,其一级动力学常数分别为2.88×10-3s-1和1.74×10-3s-1.PP2B可使磷酸化的tau蛋白脱磷酸化,并且脱磷酸化作用随肝素浓度的增加而增强,提示肝素可能具有调节tau蛋白磷酸化状态的作用     

Tau function and dysfunction in neurons: its role in neurodegenerative disorders

Alzheimer’s disease (AD) is the most usual neurodegenerative disorder leading to dementia in the aged human population. It is characterized by the presence of two main brain pathological hallmarks: senile plaques and neurofibrillary tangles (NFTs). NFTs are composed of fibrillar polymers of the abnormally phosphorylated cytoskeletal protein tau.     

Regulation of Phosphorylation of tau by Protein Kinases in Rat Brain

Microtubule associated protein tau is abnormally hyperphosphorylated in Alzheimer disease (AD) brain. To investigate the role of protein kinases involved in this lesion, metabolically active slices made from brains of adult rats were treated with or without various specific kinase activators in oxygenated artificial cerebrospinal fluid. The basal kinase activities of protein kinase-A (PKA), CaM Kinase II and GSK-3 were stimulated more than two-fold by isoproterenol, bradykinin and wortmannin, respectively. We found that cdk5 activity was co-stimulated with PKA by isoproterenol. Sequential activation of PKA (+cdk5), CaM Kinase II and GSK-3 produced hyperphosphorylation of tau at Ser-198/Ser-199/Ser-202, Ser-214, Thr-231/Ser-235, Ser-262, Ser-396/Ser-404 and Ser-422 sites. Like AD P-tau, the P-tau from brain slices bound to normal tau and its binding to tubulin was inhibited. These studies suggest that PKA, cdk5, CaM Kinase II and GSK-3 are involved in the regulation of phosphorylation of tau and that AD-type phosphorylation of tau is probably a product of the synergistic action of two or more of these kinases.     

Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5

Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.     

Prostate-derived Sterile 20-like Kinases (PSKs/TAOKs) Phosphorylate Tau Protein and Are Activated in Tangle-bearing Neurons in Alzheimer Disease

In Alzheimer disease (AD), the microtubule-associated protein tau is highly phosphorylated and aggregates into characteristic neurofibrillary tangles. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) 1 and 2, members of the sterile 20 family of kinases, have been shown to regulate microtubule stability and organization. Here we show that tau is a good substrate for PSK1 and PSK2 phosphorylation with mass spectrometric analysis of phosphorylated tau revealing more than 40 tau residues as targets of these kinases. Notably, phosphorylated residues include motifs located within the microtubule-binding repeat domain on tau (Ser-262, Ser-324, and Ser-356), sites that are known to regulate tau-microtubule interactions. PSK catalytic activity is enhanced in the entorhinal cortex and hippocampus, areas of the brain that are most susceptible to Alzheimer pathology, in comparison with the cerebellum, which is relatively spared. Activated PSK is associated with neurofibrillary tangles, dystrophic neurites surrounding neuritic plaques, neuropil threads, and granulovacuolar degeneration bodies in AD brain. By contrast, activated PSKs and phosphorylated tau are rarely detectible in immunostained control human brain. Our results demonstrate that tau is a substrate for PSK and suggest that this family of kinases could contribute to the development of AD pathology and dementia.     

Phosphorylated tau and the neurodegenerative foldopathies

Many studies have implicated phosphorylated tau in the Alzheimer disease process. However, the cellular fate of phosphorylated tau has only recently been described. Recent work has shown that tau phosphorylation at substrate sites for the kinases Cdk5 and GSK3-beta can trigger the binding of tau to the chaperones Hsc70 and Hsp27. The binding of phosphorylated tau to Hsc70 implied that the complex may be a substrate for the E3 ligase CHIP and this possibility was experimentally verified. The presence of this system in cells suggests that phosphorylated tau may hold toxic dangers for cell viability, and the response of the cell is to harness a variety of protective mechanisms. These include binding to chaperones, which may prevent more toxic conformations of the protein, ubiquitination which will direct the protein to the proteasome, segregation of tau aggregates from the cellular machinery, and recruitment of Hsp27 which will confer anti-apoptotic properties to the cell.