【作者】 朱海丽；

【导师】 梁毅；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 阿尔茨海默病的重要病理特征之一就是主要由微管结合蛋白Tau所形成的神经纤维缠结。由于Tau蛋白和硫酸乙酰肝素在神经细胞中是共定位的,因此为了模拟体内硫酸乙酰肝素介导的Tau蛋白错误折叠,我们在体外实验中加入了一种多聚阴离子-肝素-来诱导Tau蛋白形成纤维,定量研究了人Tau蛋白微管结合核心区片段Tau244-372与肝素的相互作用。研究结果表明,在生理还原环境中,肝素与Tau244-372形成紧密结合的1：1复合物,而在病理氧化环境中,肝素与Tau244-372形成中等强度结合的1：1复合物。对人Tau蛋白错误折叠过程的动力学分析表明,当肝素浓度与Tau蛋白浓度相等时,Tau蛋白纤维的生长速率最快,而在肝素浓度超过Tau蛋白浓度的条件下,Tau蛋白纤维的生长速率随着肝素浓度的增加而逐渐降低。实验结果表明,一方面,肝素与Tau蛋白单体形成1：1复合物,诱导Tau蛋白发生构象变化,进而促进Tau蛋白的错误折叠；另一方面,过量的肝素通过提高介质的离子强度来抑制Tau蛋白的错误折叠。该研究结果将有助于阐释肝素诱发Tau蛋白错误折叠的分子机制以及阿尔茨海默病的发病机制。铅离子对人、特别是对发育中的儿童而言具有强烈毒性,在阿尔茨海默病患者脑中就发现了高浓度的铅离子。然而,铅离子对神经纤维缠结形成的作用机制以及对阿尔茨海默病发病的影响还不清楚。因此我们研究了铅离子对人Tau蛋白微管结合核心区片段Tau244-372纤维化的影响。实验结果表明,在检测时间范围内5-40μM铅离子的加入不仅显著促进了疏水结构的暴露和纤维的形成,而且使Tau蛋白形成的纤维β-折叠结构含量增多。与野生型Tau蛋白不同的是,铅离子的加入对Tau蛋白组氨酸残基单突变体H362A以及双突H330A/H362A纤维形成没有明显的促进作用,也不能增加形成的纤维结构中β-折叠结构的含量。等温滴定量热实验的结果则显示,一个铅离子可以与一个Tau244-372分子以较高强度结合(Kd,0.217μM)其中330位和362位组氨酸残基是核心位点,His330和His362在铅离子促进Tau蛋白聚集的过程中起着重要的作用。我们第一次报道了铅离子通过与Tau蛋白330位和362位组氨酸残基的相互作用来促进Tau蛋白纤维的形成。该研究结果为了解铅离子对阿尔茨海默病的病理学影响提供了依据,而且有益于阐释铅离子的毒性机理。大部分蛋白质错误折叠的体外研究都在稀溶液中进行,而胞内蛋白质错误折叠则是在生理拥挤环境中发生。我们研究了生理拥挤环境中人Tau蛋白微管结合核心区片段Tau244-372及其突变体的纤维生长动力学。实验结果表明,50-150g/lFicoll70和dextran70的加入都能不同程度地促进Tau蛋白的纤维形成,且促进程度随着大分子拥挤试剂浓度的升高而提高。我们发现大分子拥挤促进Tau蛋白纤维形成的机理之一是大分子拥挤促进了Tau蛋白分子间二硫键的形成。大分子拥挤的这种促进作用对Tau蛋白的病理突变体同样适用。因此,生理拥挤环境可能通过促进Tau蛋白分子间二硫键的形成来促进和调控Tau蛋白纤维的形成,在阿尔茨海默病的病理学进程中发挥着重要的作用。更多还原

【Abstract】 Neurofibrillary tangles, principally composed of bundles of filaments formed by microtubule associate protein Tau, is a hallmark in a group of neurodegenerative diseases such as Alzheimer’s disease. Polyanionic cofactors such as heparin could induce Tau filament formation. Here we quantitatively characterize the interaction between recombinant human Tau fragment Tau244-372and heparin (average MW=7kDa) as well as heparin-induced fibril formation by using static light scattering, isothermal titration calorimetry, turbidity assays and transmission electron microscopy. Our data clearly show that at physiological pH, heparin and human Tau244-372form a tight1:1complex with an equilibrium association constant exceeding106M"1under reducing conditions, triggering Tau fibrillization. Heparin shows a moderate binding affinity (105M’1) to Tau244-372in the absence of dithiolthreitol, triggering Tau fibrillization too. Further fibrillization kinetics analyses show that the lag time appears to be approximately invariant up to a molar ratio of2:1, and then gets longer at larger ratios of heparin/Tau. The maximum slope representing the apparent rate constant for fibril growth increases sharply with substoichiometric ratios of heparin/Tau, and then decreases to some extent with ratios>1:1. The retarding effect of heparin in excess is due to the large increase in ionic strength of the medium arising from free heparin. Together, these results suggest that the formation of1:1complex of Tau and heparin plays an important role in the inducer-mediated Tau filament formation, providing clues to understanding the pathogenesis of neurodegenerative diseases.Lead is a potent neurotoxin for human being especially for the developing children, and Pb2+at high concentrations is found in the brains of patients with Alzheimer disease. However, it has not been reported so far whether Pb2+plays a role in the pathology of Alzheimer disease through interaction with human Tau protein and thereby mediates Tau filament formation. In this study, we have investigated the effect of Pb2+on fibril formation of recombinant human Tau fragment Tau244-372and its mutants at physiological pH. As revealed by thioflavin T and8-anilino-1-naphthalene sulfonic acid fluorescence, the addition of5-40μM Pb2+significantly accelerates the exposure of hydrophobic region and filament formation of wild-type Tau244-372on the investigated time scale. As evidenced by circular dichroism and Fourier transform infrared spectroscopy, fibrils formed by wild-type Tau244-372in the presence of5-40μM Pb2+contain more β-sheet structure than those formed by the protein in the absence of Pb2+. However, unlike wild-type Tau244-372, the presence of5-40μM Pb2+has no obvious effects on fibrillization kinetics of single mutants H330A and H362A and double mutant H330A/H362A, and fibrils formed by such mutants in the absence and in the presence of Pb2+contain similar amounts of β-sheet structure. The results from isothermal titration calorimetry show that one Pb+binds to one Tau monomer via interaction with His-330and His-362, with sub-micromolar affinity. We demonstrate for the first time that the fibrillization of human Tau protein is accelerated by exposure to lead via interaction with His-330and His-362. Our results suggest the possible involvement of Pb+in the pathogenesis of Alzheimer disease and provide critical insights into the mechanism of lead toxicity.The role of crowded intracellular environments on Tau protein misfolding is not clearly understood. The addition of Ficoll70and dextran70at50-150g/1significantly accelerates filament formation of Tau244-372on the investigated time scale, and the enhancing effects increase with the increase of concentrations of crowding agents. Our data demonstrate that macromolecular crowding enhances the formation of inter-molecular disulfide bond between C291and C322of Tau244-372.Such an enhancing effect is also observed for pathological Tau mutants. Our data demonstrate that a crowded physiological environment could play an important role in the pathogenesis of Alzheimer disease by enhancing the formation of inter-molecular disulfide bond of Tau protein.更多还原