蛋白激酶与阿尔采末病

阿尔采末病（Ａｌｚｈｅｉｍｅｒ‘ｓ ｄｉｓｅａｓｅ,ＡＤ）的典型病症之一是学习和记忆功能障碍。老年斑,淀粉样蛋白沉积及神经原纤维缠结（ＮＦＴ）是其大脑主要的病理学特征。动物在学习和记忆行为实验时记忆的获得与保持以及海马长时程增强效应（ＬＴＰ）的诱生均与蛋白激酶Ｃ（ＰＫＣ）的活性呈明显的正相关,蛋白激酶可能通过信号转导途径调节β－淀粉样蛋白的前体蛋白剪切代城Ａβ,ｔａｕ蛋白的磷酸化是ＮＦＴ形成的主要     

蛋白激酶A与阿尔茨海默病

阿尔茨海默病 (Ａｌｚｈｅｉｍｅｒｄｉｓｅａｓｅ ,ＡＤ)是老年痴呆症中最常见的一种 ,主要表现为进行性的记忆和认知功能障碍。目前ＡＤ的病因和发病机制尚不明了。神经原纤维缠结(ｎｅｕｒｏｆｉｂｒｉｌｌａｒｙｔａｎｇｌｅ ,ＮＦＴ)作为ＡＤ的特征性病变 ,与临床痴呆症状呈正相关 ,提示ＮＦＴ在ＡＤ的发病过程中可能发挥重要作用[1] 。ＮＦＴ以成对螺旋丝样结构 (ｐａｉｒｅｄｈｅｌｉｃａｌｆｉｌａ ｍｅｎｔ,ＰＨＦ)存在 ,其主要成分是异常过度磷酸化的ｔａｕ蛋白 (ｔａｕｐｒｏｔｅｉｎ) [2 ] 。Ｔａｕ蛋白在成人脑中有 6种同分异…     

Alzheimer病脑内发现第三种病理损害

Ａｌｚｈｅｉｍｅｒ病脑内发现第三种病理损害多年来,研究人员一直从β淀粉样蛋白和ｔａｕ蛋白着手寻找ＡＤ治疗的新线索。最近在ＡＤ脑内发现了一种新的病理损害“ＡＭＹ斑块”,其在ＡＤ脑内的分布与β淀粉样蛋白斑块和ｔａｕ蛋白缠结一样广泛,表明ＡＭＹ斑块可能...     

蛋白磷酸酯酶-1和-2A抑制剂对NG108-15细胞tau蛋白磷酸化的影响

Alzheimer痴呆(AD)的主要脑病理变化之一为由超磷酸化的tau蛋白组成的神经原纤维缠结(Neurofibrillary tangle,NFT)。AD的Tau蛋白异常磷酸化与蛋白磷酸酯酶(PP)-2A和-1缺陷有关。本文首先用免疫印迹法显示NG含两大组分的tau蛋白。MTT方法观察到PP-2A和PP-1抑制剂冈 田酸(Okadaic acid.OA)处理NG108-14细胞能导致细胞代谢明显下降,同时免疫印迹法显示OA能导致的NG细胞Tau蛋白磷酸化。该研究为建立AD样蛋白磷酸酯酶缺陷引起的tau蛋白磷酸化的细胞模型奠定了基础。     

神经生长抑制因子与阿尔茨海默病

阿尔茨海默病 (Ａｌｚｈｅｉｍｅｒ′ｓｄｉｓｅａｓｅ ,ＡＤ)是一种以进行性认知障碍和记忆能力损害为主的中枢神经系统退行性疾病。其病理特征为神经元内出现大量双股螺旋纤丝 (ｐａｉｒｅｄｈｅｌｉｘｆｉｌａｍｅｎｔ,ＰＨＦ)和细胞外间隙出现 β 淀粉样蛋白 (β ａｍｙｌｏｉｄ ,Ａβ)的沉积 ,即神经元纤维缠结 (ｎｅｏｒｏｆｉｂｒｉｌｌａｒｙｔａｎｇｌｅｓ,ＮＦＴ)和老年斑(ｓｅｎｉｌｅｐｌａｑｕｅ,ＳＰ)的大量存在以及选择性的神经元和突触的丢失[1] 。ＡＤ可分为家族性 (ＦＡＤ)和散发性 (ＳＡＤ)。神经生长抑制因子 (ｎｅｕｒ…     

蛋白酶体与神经原纤维缠结

蛋白酶体在真核生物体内选择性识别、清除错误折叠和异常聚积的蛋白质。神经原纤维缠结是阿尔茨海默病患典型的病理特征之一,主要由异常磷酸化的微管相关蛋白tau聚积而成,但其形成机制尚未阐明。越来越多的研究表明蛋白酶体功能异常和神经原纤维缠结的形成密切相关。     

Tau蛋白——Alzheimer病研究的新热点

Ｔａｕ蛋白——Ａｌｚｈｅｉｍｅｒ病研究的新热点顾琪贺林（上海生命科学研究中心,上海２０００３１）关键词Ｔａｕ蛋白Ａｌｚｈｅｉｍｅｒ病Ｔａｕ蛋白是一种神经原微管相关蛋白。处于异常磷酸化状态的Ｔａｕ蛋白集合成双股螺旋纤丝（ｐａｉｒｅｄｈｅｌｉｃａｌｆｉｌ...     

酵母PH02蛋白的磷酸化研究

本文报道ＰＨＯ２蛋白能被一种未知的蛋白激酶磷酸化ＰＨＯ２蛋白的第２３０－２３３位氨基酸残基组成一个可能被ｐ３４相关的蛋白激酶识别的一致序列（ＳＰＩＫ）,用点突变的方法将Ｓｅｒ－２３０变成Ａｌａ可导致ＰＨＯ２蛋白激活ＰＨＯ５表达能力的完全丧失。进一步的研究显示,将Ｐｒｏ－２３１突变成Ｓｅｒ同样可导致ＰＨＯ２的矢活,而Ｓｅｒ－２３０突变成Ａｓｐ则不影响ＰＨＯ２的活力。由于ＰＨＯ２（Ａｓｐ－２３０）突变体通过在第２３０位残基引入了一个负电荷,可以看成Ｓｅｒ－２３０被磷酸化的状态,由此推测ＰＨＯ２蛋白可能需要在Ｓｅｒ－２３０被磷酸化后才会具有激活ＰＨＯ５基因转录的能力。体外磷酸化分析的结果表明,大肠杆菌表达的ＧＳＴＩ－ＰＨＯ２（野生型）融合蛋白能够被酵母ＹＰＨ４９９总蛋白抽提物磷酸化,如果以ＳＰＩＫ（２３０－２３３）一致序列被破坏的ＧＳＴ－ＰＨＯ２（Ｐｒｏ－２３１→Ｓｅｒ）突变体作为底物,则观察不到该融合蛋白被酸磷化标记。结果表明ＰＨＯ２在细胞内是一种磷骏化蛋白,第２３０位Ｓｅｒ的磷酸化是该转录因子较制ＰＨＯ５基因表达的活性所必需。     

荧光指示剂法测定植物细胞胞质游离Ca^2＋浓度

利用钙离子荧光指示剂Ｉｎｄｏ－１ ＡＭ 建立了测定植物细胞胞质游离Ｃａ２＋ 浓度的技术。应用此技术测出,ＢＭＳ（ｂｌａｃｋ Ｍｅｘｉｃｏ ｓｗ ｅａｔ）玉米悬浮细胞原生质体静息水平下的胞质游离Ｃａ２＋ 浓度是１２７±５６ ｎｍ ｏｌ·Ｌ－ １;Ｃａ２＋ 螯合剂ＥＧＴＡ 可使胞质游离Ｃａ２＋ 浓度由７８ ｎｍ ｏｌ·Ｌ－ １降至１２．５ ｎｍ ｏｌ·Ｌ－ １,而Ｃａ２＋ 载体Ａ２３１８７ 则可使胞质游离Ｃａ２＋ 浓度升至接近介质Ｃａ２＋ 水平。同时,证明ＡＢＡ 处理可使ＢＭＳ玉米悬浮细胞原生质体Ｃａ２＋ 浓度在１—１．５ 分钟内迅速升高,由７５ ｎｍ ｏｌ·Ｌ－ １升至７９０ ｎｍ ｏｌ·Ｌ－ １     

DDPH对猪肺动脉平滑肌细胞PDGF·BB和bFGF表达的影响：免疫细胞化学研究

ＤＤＰＨ［１－（２．６－二甲基苯乙氧基）－２－（３．４二甲氧基苯乙胺基）丙烷盐酸盐］是南京药科大学合成的降压新化合物,也具有降低肺动脉高压和抑制肺动脉平滑肌细胞增殖作用。本实验用细胞培养、免疫细胞化学、图像分析、３Ｈ－ＴｄＲ、细胞周期测定等方法,进一步探讨ＤＤＰＨ对缺氧性肺动脉平滑肌细胞（ＰＡＳＭＣＳ）增殖的抑制机制。结果：缺氧促进肺动脉内皮细胞（ＰＡＥＣｓ）的ＰＤＧＦ·ＢＢ和ｂＦＧＦ两种生长因子的表达（积分光密度ＯＤ值）增高。缺氧内皮细胞条件培养液（ＨＥＣＣＭ）能促进ＰＡＳＭＣＳ的ＰＤＧＦ·ＢＢ的ＯＤ值增高,ｂＦＧＦ的ＯＤ值无明显改变。加药组（ＨＥＣ－ＣＭ＋ＤＤＰＨ）的ＰＤＧＦ·ＢＢ和ｂＦＧＦ的ＯＤ值均显著降低,尤以ＰＤＧＦ·ＢＢ的ＯＤ值减少最多．提示：ＤＤＰＨ能抑制ＨＥＣＣＭ引起ＰＡＳＭＣＳ的ＰＤＧＦ·ＢＢ和ｂＦＧＦ表达增多和细胞增殖。结果与大鼠实验观察相符。     

Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease

The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation.     

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     

Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology

Down-regulation of protein phosphatase 2A (PP2A) is thought to play a critical role in tau hyperphosphorylation in Alzheimer's disease (AD). In vitro phosphorylation of PP2A catalytic subunit at Y307 efficiently inactivates PP2A. A specific antibody against phosphorylated (p) PP2A (Y307) (PP2Ac-Yp307) was used to investigate possible PP2A down-regulation by known pathophysiological changes associated with AD, such as Abeta accumulation and oestrogen deficiency. Immunohistochemistry and immunofluorescence confocal microscopy showed an aberrant accumulation of PP2Ac-Yp307 in neurons that bear pretangles or tangles in the susceptible brain regions, such as the entorhinal cortical cortex and the hippocampus. Experimentally, increased PP2Ac-Yp307 was observed in mouse N2a neuroblastoma cells that stably express the human amyloid precursor protein with Swedish mutation (APPswe) compared with wild-type, and in the brains of transgenic APPswe/ presenilin (PS1, A246E) mice, which corresponded to the increased tau phosphorylation. Treating N2a cells with Abeta25-35 mimicked the changes of PP2Ac-Yp307 and tau phosphorylation in N2a APPswe cells. Knockout of oestrogen receptor (ER) alpha or ERbeta gave similar changes of PP2Ac-Yp307 level and tau phosphorylation in the mouse brain. Taken together, these findings suggest that increased PP2A phosphorylation (Y307) can be mediated by Abeta deposition or oestrogen deficiency in the AD brain, and consequently compromise dephosphorylation of abnormally hyperphosphorylated tau, and lead to neurofibrillary tangle formation.     

Phosphorylation of tau at Ser214 mediates its interaction with 14-3-3 protein: implications for the mechanism of tau aggregation

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine –phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro . The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.     

Dephosphorylation of tau protein by calcineurin triturated into neural living cells

Alzheimer disease and related dementia are characterized by the presence of hyperphosphorylated tau aggregated into filaments. The role of tau phosphorylation in the fibrillogenesis has not yet been unraveled. Therefore, it is important to know which phosphatases can dephosphorylate tau protein in vivo. The effect of recombinant purified calcineurin (CN(PP2B)) and several calcineurin mutants on tau phosphorylation was studied in two neuronal like cell lines PC12 and SH-SY5Y. The modulation of tau phosphorylation at Ser199/Ser202, Ser396/Ser404, Ser262/Ser356, and Thr181 sites was examined in these cell lines using the phosphorylation state-dependent antitau antibodies Tau 1, PHF1, 12E8, and AT270. The results have shown that CN directly dephosphorylates all of those sites of tau protein. Recombinant calcineurin introduced into cells that have previously been treated with okadaic acid and cyclosporin A, which are inhibitors of phosphatases (PP1/PP2A and PP2B), has a direct effect on the phosphorylation status on all phosphorylation sites studied. We conclude that calcineurin is (besides PP2A) a important modulator of tau phosphorylation in vivo.     

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.     

Dephosphorylation of tau by protein phosphatase 5: impairment in Alzheimer's disease

Protein phosphatase (PP) 5 is highly expressed in the mammalian brain, but few physiological substrates have yet been identified. Here, we investigated the kinetics of dephosphoryation of phospho-tau by PP5 and found that PP5 had a K(m) of 8-13 microm toward tau, which is similar to that of PP2A, the major known tau phosphatase. This K(m) value is within the range of intraneuronal tau concentration in human brain, suggesting that tau could be a physiological substrate of both PP5 and PP2A. PP5 dephosphorylated tau at all 12 Alzheimer's disease (AD)-associated abnormal phosphorylation sites studied, with different efficiency toward each site. Thr(205), Thr(212), and Ser(409) of tau were the most favorable sites; Ser(199), Ser(202), Ser(214), Ser(396), and Ser(404) were less favorable sites; and Ser(262) was the poorest site for PP5. Overexpression of PP5 in PC12 cells resulted in dephosphorylation of tau at multiple phosphorylation sites. The activity but not the protein level of PP5 was found to be decreased by approximately 20% in AD neocortex. These results suggest that tau is probably a physiological substrate of PP5 and that the abnormal hyperphosphorylation of tau in AD might result in part from the decreased PP5 activity in the diseased brains.     

Involvement of I2PP2A in the abnormal hyperphosphorylation of tau and its reversal by Memantine

The activity of protein phosphatase (PP)-2A, which regulates tau phosphorylation, is compromised in Alzheimer disease brain. Here we show that the transient transfection of PC12 cells with inhibitor-2 (I2PP2A) of PP2A causes abnormal hyperphosphorylation of tau at Ser396/Ser404 and Ser262/Ser356. This hyperphosphorylation of tau is observed only when a sub-cellular shift of I2PP2A takes place from the nucleus to the cytoplasm and is accompanied by cleavage of I2PP2A into a 20 kDa fragment. Memantine, an un-competitive inhibitor of N-methyl-D-aspartate receptors, inhibits this abnormal phosphorylation of tau and cell death and prevents the I2PP2A-induced inhibition of PP2A activity in vitro. These findings demonstrate novel mechanisms by which I2PP2A regulates the intracellular activity of PP2A and phosphorylation of tau, and by which Memantine modulates PP2A signaling and inhibits neurofibrillary degeneration.     

The protein phosphatase PP2A/Bα binds to the microtubule-associated proteins Tau and MAP2 at a motif also recognized by the kinase Fyn: implications for tauopathies

The predominant brain microtubule-associated proteins MAP2 and tau play a critical role in microtubule cytoskeletal organization and function. We have previously reported that PP2A/Bα, a major protein phosphatase 2A (PP2A) holoenzyme, binds to and dephosphorylates tau, and regulates microtubule stability. Here, we provide evidence that MAP2 co-purifies with and is dephosphorylated by endogenous PP2A/Bα in bovine gray matter. It co-localizes with PP2A/Bα in immature and mature human neuronal cell bodies. PP2A co-immunoprecipitates with and directly interacts with MAP2. Using in vitro binding assays, we show that PP2A/Bα binds to MAP2c isoforms through a region encompassing the microtubule-binding domain and upstream proline-rich region. Tau and MAP2 compete for binding to and dephosphorylation by PP2A/Bα. Remarkably, the protein-tyrosine kinase Fyn, which binds to the proline-rich RTPPKSP motif conserved in both MAP2 and tau, inhibits the interaction of PP2A/Bα with either tau or MAP2c. The corresponding synthetic RTPPKSP peptide, but not the phosphorylated RpTPPKSP version, competes with Tau and MAP2c for binding to PP2A/Bα. Significantly, down-regulation of PP2A/Bα and deregulation of Fyn-Tau protein interactions have been linked to enhanced tau phosphorylation in Alzheimer disease. Together, our results suggest that PP2A/Bα is part of segregated MAP2 and tau signaling scaffolds that can coordinate the action of key kinases and phosphatases involved in modulating neuronal plasticity. Deregulation of these compartmentalized multifunctional protein complexes is likely to contribute to tau deregulation, microtubule disruption, and altered signaling in tauopathies.