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。     

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。     

Tau蛋白在阿尔兹海默病治疗中的研究进展

阿尔兹海默病中一个重要的病理特点是神经原纤维缠结(NFTs),其与Tau蛋白具有密切的关系。Tau蛋白是含量最高的微管相关蛋白。正常Tau蛋白可与微管蛋白结合促进其有效聚合形成微管。而过度磷酸化的Tau蛋白则会自我聚集,进而导致NFTs的形成。近年来,国内外就Tau为主要靶点治疗AD有了很大进展。本文就Tau蛋白在AD中的病理学意义,与Aβ蛋白的相互作用以及以Tau为主要切入点对AD进行治疗的方法作一综述。     

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...     

早老蛋白与Alzheimer病

遗传因素是引起Alzheimer病的原因之一,其中的早发型病例大多数是由早老蛋白－1和早老蛋白－2的基因突变引起的。这两个基因分别位于第1和14号染色体上,两者的氨基酸序列具有很高的同源性,均含7次疏水跨膜结构。对两者突变致病机制的研究可能是解开Alzheimer病发病之谜的钥匙。此外,早老蛋白－1、－2可能参与从细胞膜到核的信息传递,调节细胞的生长、分化、凋亡等。     

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。     

Alzheimer氏病淀粉样前体蛋白的研究进展

Alzheimer氏病(AD)是一种发生于老年人群的原发性退行性脑病,其特征性病变为细胞内神经纤维缠绕(NFT)及细胞外老年斑(SP).构成SP的主要成分β淀粉样多肽(βA)为一由淀粉样前体蛋白(APP)剪切而来的分子质量约为4 ku的多肽,其神经毒性可能由其氧化作用和在脂质双层中形成的Ca²⁺通道所致.APP的功能目前尚未完全明了,可能具有促进细胞粘附、维护突触膜稳定性等功能.APP主要通过两种途径进行加工修饰：一为分泌途径,由一些假定的分泌酶催化;另一为胞内体-溶酶体途径.在形成SP的βA中,较长者比短者更易聚集,因此一些APP突变由于能够释放出更多的较长的βA或者使较短的βA生成量增加而致发家族性AD.一些可能在APP的代谢中起着重要作用的因素,如早衰蛋白的突变,也可通过增加βA的生成量而致发AD.     

铝参与神经原纤维缠结形成的实验研究

目的探讨铝与神经原纤维缠结(NFTs)形成之间的相关性。方法选用16只雌性ICR小鼠,分为正常对照组与染铝组(200mg/kg.bw,染铝8个月)。组织荧光双重染色法观察铝与NFTs在小鼠大脑新皮层神经元内的定位。West-ern blot法半定量检测新皮层内tau蛋白及其磷酸化水平。结果组织荧光染色表明NFTs阳性荧光表达随铝阳性荧光增强而增强,两者分布呈对应关系;Western blot结果显示长期铝暴露导致tau蛋白水平下降(P<0.01,与对照组相比),但磷酸化水平升高(P<0.01,与对照组相比)。结论铝参与大脑新皮层神经元内NFTs形成与累积过程,tau蛋白的过度磷酸化可能是其成因之一。     

蛋白磷酸酯酶2A调节微管相关蛋白1b的磷酸化

微管相关蛋白MAP1b的生物学活性受其磷酸化修饰的调节,后者则受相应的蛋白激酶和蛋白磷酸酯酶(PP)调控.为研究蛋白磷酸酯酶在脑内对MAP1b磷酸化的调控作用,采用有代谢活性的大鼠脑片作为模型,分别应用冈田酸(okadaic acid)和cyclosporin A选择性地抑制PP2A 和PP2B活性,来研究其对脑内蛋白磷酸酯酶MAP1b磷酸化的调控.采用特异性的MAP1bⅠ型磷酸化依赖性抗体522和免疫印迹技术检测MAP1bⅠ型磷酸化.结果表明,当PP2A被okadaic acid选择性抑制后,MAP1bⅠ型磷酸化明显增加.而PP2B被选择性地抑制后,MAP1b磷酸化的变化不大.免疫组化染色显示,MAP1b广泛分布于鼠大脑神经元和突起中,与对照组相比,在PP2A抑制的脑片中抗体522的免疫活性在神经元中明显升高.上述结果表明,PP2A是脑中调控MAP1bⅠ型磷酸化的主要蛋白磷酸酯酶.     

酿酒酵母丝氨酸/苏氨酸蛋白磷酸酯酶的功能与调控

从酿酒酵母蛋白磷酸酯酶的分类和结构特征入手,阐述了该蛋白家族中的亚家族成员丝氨酸/苏氨酸蛋白磷酸酯酶的功能和表达调控.深入研究酿酒酵母丝氨酸/苏氨酸蛋白磷酸酯酶,特别是PP2C蛋白磷酸酯酶的细胞功能及其调控,将对新药研发和疾病干预治疗提供重要基础.     

Characterization of the Glycolipid Associated with Alzheimer Paired Helical Filaments

Abstract: In the present study, analytical techniques including gas chromatography/mass spectrometry (GC/MS)-assisted carbohydrate linkage-analysis, one- and two-dimensional NMR, and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-MS) have been used to characterize the structure of the glycolipid associated with the paired helical filaments (PHF) isolated from the neurofibrillary tangles of Alzheimer's diseased brain. The ¹H NMR spectrum of acid-hydrolyzed protein-resistant core PHF (prcPHF) displays resonances that can be assigned to fatty acid and glucose. There are no resonances present that would indicate the presence of protein, amino acids, or a sphingosine base. Using two-dimensional homonuclear correlated spectroscopy, homonuclear Hartmann-Hahn, and heteronuclear multiple quantum coherence experiments, resonances in the ¹H and ¹³C NMR spectrum of native PHF were assigned to a nonreducing terminal α-1,6-glycosidically linked glucose, an internal α-1,6-linked glucose, and an α-1,2,6-linked glucose. The narrow line-widths observed for these residues suggest that they arise from glucose residues undergoing rapid segmental motion. The carbohydrate portion of the PHF-associated glycolipid was analyzed using GC/MS linkage analysis and confirmed the presence of terminal and internal α-1,6-linked glucose and α-1,2,6-linked glucose in a molar ratio of 2:1:1. Three components of the PHF-associated glycolipid fraction having masses 2,416, 2,325, and 2,237 Da were observed using MALDI-MS. The least abundant, heavier mass component (2,416 Da) was best fit to a structure with a tridecamer of glucose having a single esterified C₂₀ fatty acid (Glc₁₃ + C₂₀ or Glc₁₃ + C_20:1), whereas the more abundant, lower mass components were best fit to noncovalently associated glycolipid dimers, each with a glucose pentamer or hexamer having two C₁₄, C₁₆, or C₁₈ esterified fatty acids {D[(Glc₅ + C₁₈) + (Glc₆ + C₁₆)] or D[(Glc₅ + C₁₄) + (Glc₆ + C₁₄)]}. The ratio of glucose to fatty acid calculated from these best-fit structures of the more abundant mass components (5.5 ± 1.1:1.0) is in reasonable agreement with the same ratio calculated from peak integrations in the NMR spectra of acid-hydrolyzed prcPHF (6.2 ± 1.6). Structural similarities between PHF-associated glycolipid and other glycolipid amphiphiles known to form PHF-like filaments indirectly suggest that this unique glycolipid may be an integral component of the PHF suprastructure.     

Immunochemical Properties of Ubiquitin Conjugates in the Paired Helical Filaments of Alzheimer Disease

Immunocytochemical and peptide sequencing studies indicate that the regulatory protein ubiquitin (Ub) is incorporated into the paired helical filaments (PHF) of Alzheimer disease. In this study, we showed that some antibodies raised to PHF recognize epitopes of Ub. Analysis of the Ub sequences recognized by the antibodies raised to PHF, along with the known specificity of several monoclonal antibodies raised to artificial Ub conjugates, indicates the immunochemical representation of Ub residues 34-76 in PHF. The Ub epitopes recognized by antibodies raised to PHF are distinct from those recognized by antibodies raised to artificial Ub conjugates in two respects. First, antibodies that are raised to PHF and that recognize Ub react with PHF equally, whether denatured or not, whereas those raised to artificial Ub conjugates show greater reaction after denaturation. Second, mapping of the epitopes recognized by two monoclonal antibodies to PHF onto Ub indicates a distinction in the Ub residues recognized, compared with monoclonal antibodies raised to artificial Ub conjugates. The proximity of their epitopes to the site of conjugation, as well as their affinity for PHF polypeptides, suggests that the PHF antibodies that recognize Ub may be directed specifically to Ub epitopes defined by the protein conjugated to Ub.     

Purification and Solubilization of Paired Helical Filaments from Alzheimer Brains

The purpose of the present study was to develop a purification and solubilization method, compatible with current amino acid sequencing techniques, for paired helical filaments (PHFs) derived from patients with Alzheimer's disease. We have developed a mild procedure that subjects conventionally isolated PHFs to Tris/borate/sodium dodecyl sulfate/2-mercaptoethanol electrophoresis and results in the separation of the relatively insoluble PHF structures from both copurifying contaminating proteins and solubilized PHF-associated proteins. At the end of 4.5 h of electrophoresis, the purified insoluble fraction had an amino acid composition that was invariant during subsequent electrophoresis. Electron microscopy revealed an intact PHF structure before and after electrophoresis but no evidence of any other structures in the insoluble fraction, a result consistent with the removal of PHF-associated proteins from the filament structure. Isolated insoluble filament structures displayed an enhanced immunoreactivity with antibodies raised against purified PHFs in other laboratories, when compared with the fraction not subjected to electrophoresis in enzyme-linked immunosorbent assays. Solubilization of the relatively insoluble PHFs was accomplished by extending the time of electrophoresis beyond the 4.5 h required for purification. Additional electrophoresis for 34.5 h solubilized 88% of the purified, relatively insoluble PHFs. This resulted in the identification of four major protein bands between Mr values of approximately 50,000 and 70,000 on sodium dodecyl sulfate-polyacrylamide electrophoresis gel analysis, with a predominant band with an Mr of approximately 66,000. A slow fragmentation of the PHF ultrastructure occurred during this time, as judged by electron microscopy. This purification technique will permit the isolation of consistently reproducible protein fragments from solubilized PHFs, which may be used for subsequent sequence analysis.     

A τ-Related Protein of 130 kDa Is Present in Alzheimer Brain

Two abnormal entities of 69 and 130 kDa, immunologically related to the microtubule-associated tau proteins, are present in the hippocampus and the frontal cortex of the Alzheimer brain, which contain a large number of neurofibrillary tangles (NFTs), but are absent in the cerebellum, which does not contain these structures. Epitope mapping with antibodies spanning domains present in the N-terminal, middle, and C-terminal tau sequence demonstrated that the 69- and 130-kDa entities belong to the tau family. Both the 69- and the 130-kDa proteins were found in an insoluble form and were the major tau species present in purified NFTs. A procedure was devised that allowed us to prepare from Alzheimer hippocampi two NFT fractions differing in size (20 and 3 microns), both of which contained the tau entities of 130 and 69 kDa.     

Endogenous Basic Protein Phosphatases in the Brain Myelin

Direct treatment of brain myelin with freezing/thawing in 0.2 M 2-mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P-labeled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X-100, this endogenous latent phosphatase activity was further stimulated 2.5-fold. Diethylaminoethyl-cellulose and Sephadex G-200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M 2-mercaptoethanol and with a molecular weight of 350,000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP X Mg-dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high-molecular-weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.     

Inactivation and Reactivation of the Multifunctional Calmodulin-Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain

The multifunctional calmodulin-dependent protein kinase (calmodulin-kinase) from rat brain was autophosphorylated in a Ca2+- and calmodulin-dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin-kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin-kinase were universally dephosphorylated by protein phosphatase C. Calmodulin-kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin-kinase by autophosphorylation and dephosphorylation was observed using calmodulin-kinase from canine heart. These results suggest that the activity of calmodulin-kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin-kinases in various tissues.     

Characterization by Atomic Force Microscopy of Alzheimer Paired Helical Filaments under Physiological Conditions

Paired helical filaments (PHF) is an aberrant structure present in the brain of Alzheimer's disease patients which has been correlated with their degree of dementia. In order to determine the structure of PHF, several studies have been performed using atomic force microscopy (AFM). However, those studies have the limitation that they have not been done in solution and the sample could be far from the real physiological conditions. In this work we present an AFM analysis of PHF in liquid environment and we compare that analysis with that performed in dry conditions. PHF imaging in liquid was only possible by using jumping mode AFM as the imaging technique. Jumping mode AFM images of PHF in solution show first, a notable increase in the absolute values of the height of the filament, and second, a smaller ratio between the height measured at the upper and at the lower part of the PHF. Direct comparison of the experimental data with structural models has been performed. From this we conclude that the PHF structure is compatible with two coupled ribbons with an overall height of 20 nm and a width of 10 nm.     

Casein Kinase 1 Is Tightly Associated with Paired-Helical Filaments Isolated from Alzheimer's Disease Brain

Abstract: The protein kinase activity tightly associated with paired helical filaments (PHFs) purified from the brain tissue of individuals with Alzheimer's disease has been characterized in vitro. The activity is shown to phosphorylate casein, an exogenous substrate, with a maximal velocity of ∼2 nmol/min/mg, suggesting it comprises a significant component of the total protein in the PHF preparation. On the basis of substrate selectivity, isoquinoline sulfonamide inhibitor selectivity, in-gel renaturation assays, and western analysis, the activity consists of closely related members of the α branch of the casein kinase 1 family of protein kinases. Because of its tight association with PHFs and its phosphate-directed substrate selectivity, casein kinase 1 is positioned to participate in the pathological hyperphosphorylation of tau protein that is observed in neurodegenerative diseases such as Alzheimer's disease.     

Characterization of Two Distinct Monoclonal Antibodies to Paired Helical Filaments: Further Evidence for Fetal-Type Phosphorylation of the τ in Paired Helical Filaments

Abstract: Two monoclonal antibodies C5 and M4 raised against Sarkosyl-insoluble paired helical filaments (PHF) specifically labeled fetal τ, but hardly labeled normal adult τ. C5 immunoreactivity was eliminated by alkaline phosphatase treatment at 37°C, whereas M4 reactivity could be removed only by the treatment at 67°C. Epitope analysis showed that C5 and M4 recognition sites are in residues 386–406 and 198–250, respectively, according to the numbering of the longest human τ isoform. Thus, the phosphorylation sites are located in the amino- and carboxyl-terminal portions of the microtubule-binding region. These two well-characterized monoclonals should be valuable in the identification of a protein kinase(s) that converts normal τ into PHF-τ.