【作者】 蒋伟；

【导师】 刘长林；

【作者基本信息】 华中科技大学 ， 无机化学， 2007， 博士

【摘要】 肌萎缩侧索硬化症(ALS)是一种发病机理未知的较常见成人神经退行性疾病。铜锌超氧化物歧化酶(SOD1)突变产生某种未知的获得性新功能被认为是导致ALS的原因之一,然而目前并不清楚SOD1在ALS发病过程中的作用。核酸作为贮存、传递遗传信息的物质,在生命活动中发挥极为重要的作用,轻微的损伤也会影响生物体的正常功能。由于目前已发现DNA损伤涉及某些神经退性行疾病,因此,本文从SOD1与DNA相互作用的角度,探讨SOD1的获得性新功能,取得如下主要结果:1.利用紫外-可见吸收光谱和SOD1内源荧光猝灭的方法测定了SOD1对DNA的亲和性,利用琼脂糖凝胶电泳研究了SOD1和apoSOD1在二价金属离子存在下将超螺旋质粒DNA断裂为缺口形式和线性形式,以及将线性DNA断裂为小片段的性质,结果表明在外源二价金属离子存在下SOD1具有断裂DNA的核酸酶活性。稳态动力学实验结果显示SOD1和apoSOD1断裂DNA符合米氏动力学规律。与其他蛋白质和酶比较,发现SOD1这种核酸酶活性具有相对特异性,并非所有蛋白质都具有这种性质。SOD1的这种核酸酶活性是其固有的性质,与催化超氧阴离子歧化的活性中心无关。2.利用生物化学方法研究了外源二价金属离子在SOD1核酸酶活性中所起的作用。基于“非等价多部位结合”模型,我们对金属离子滴定SOD1-DNA复合物的紫外-可见吸收光谱数据进行了拟合,结果显示复合物能够提供至少两个金属结合部位:一个强结合部位和一个弱结合部位,表明SOD1可能通过“双外源金属离子”的途径断裂DNA,即两个金属离子直接参与DNA断裂过程。另外,对pH-SOD1断裂DNA速率曲线进行拟合的结果显示催化过程中存在一个一般酸和一个一般碱。结合组氨酸特异性化学修饰结果,我们提出了SOD1在二价金属离子存在下水解DNA的模型:即通过组氨酸、金属结合水分子和双金属离子三者的协同作用水解DNA。3.将正常SOD1暴露于酸性环境模拟金属结合区域SOD1突变体因突变导致的蛋白质稳定性降低、聚集倾向增强的性质,探讨了DNA加速SOD1聚集机理。利用直角光散射(RALS)、激光动态光散射(DLS)、原子力显微镜(AFM)、荧光显微镜系统研究了低pH下DNA加速SOD1聚集的性质,结果表明在酸性条件下,DNA可以作为模板加速SOD1形成聚集体。由低pH和与DNA相互作用导致的SOD1疏水性增强和DNA的模板富集效应是SOD1快速聚集的两个决定性因素。4.利用透射电镜(TEM)、AFM、荧光显微镜系统研究了酸性条件下DNA加速SOD1形成的聚集体形态。在不同的DNA浓度下形成不同类型的聚集体(聚集单体,寡聚体,大聚集体等)。SOD1与DNA的比例影响聚集体的紧密程度,比例较高时DNA形成紧密的聚集态,比例较低时则形成较为松散的聚集态。结合聚集动力学等实验结果,证实DNA加速SOD1聚集的过程也是SOD1诱导DNA凝聚的过程,提出了SOD1与DNA相互作用导致SOD1聚集和DNA凝聚的反应模型。5.利用酸性条件(pH4.0)对SOD1金属结合性质和结构稳定性的影响,模拟SOD1突变的效果,由RALS、DLS、荧光显微镜、AFM、TEM系统研究了ssDNA(24碱基)加速SOD1聚集的性质。结果表明ssDNA可以作为SOD1聚集的模板,促进纳米和微米级ssDNA-SOD1聚集单体、寡聚体、微聚集体和大聚集体快速形成。因酸性环境和ssDNA相互作用导致的SOD1疏水性增强和蛋白质局部浓度增大是SOD1快速聚集的决定性因素。ssDNA加速SOD1快速形成不溶性的大聚集体,有可能作为一种潜在的途径避免SOD1寡聚体累积产生的毒性,成为治疗或预防包括ALS在内的神经退行性疾病的药物。6.利用琼脂糖凝胶电泳、RALS、DLS、TEM系统研究了近生理条件下DNA加速被氧化SOD1聚集的性质。结果表明,当SOD1与DNA共存于氧化环境中时,DNA显著加速SOD1聚集。该结果为DNA在酸性到中性的条件下均加速SOD1聚集的假设提供了证据,即氧化导致SOD1疏水性增强和DNA的模板富集效应是SOD1快速聚集的两个决定性因素。我们推测所有SOD1突变体都可能具有被DNA加速聚集的共同性质, SOD1与DNA异常相互作用加速蛋白质聚集的性质可能是SOD1导致ALS的一种获得性新功能。更多还原

【Abstract】 Amyotrophic lateral sclerosis (ALS) is one of the most common adult neurodegenerative diseases with unknown causes. Although it is well established that SOD1 mutants involve development of ALS, and cause motor neuron death through an as-yet unidentified gain of one or more injurious properties, little is currently known about the roles of SOD1 in neurocytotoxicity. DNA is the source of genetic information and possesses many important roles in life process. Even the weak damage in DNA could have profound effect on viability and genetic stability. Moreover, DNA damage in human cells and organs has been found to be associated with neurodegenerative disease. Here, the research on the interaction between SOD1 and DNA has been carried out. The main results are as follows:1. A new activity that the SOD1 and its apo form possess a divalent metal-dependent nucleolytic activity was confirmed by UV-visible absorption titration of calf thymus DNA (ctDNA) with the SOD1, fluorescence quenching of SOD1 by ctDNA, and by gel electrophoresis monitoring conversion of DNA from the supercoiled DNA to nicked and linear forms, and fragmentation of a linearλDNA. Moreover, the DNA cleavage activity was examined in detail under certain reaction conditions. The steady state study indicates that DNA cleavage supported by both forms of SOD1 obeys Michaelis-Menten kinetics. On the other hand, the assays with some other proteins indicate that this new gain function is specific to some proteins including the SOD1. Therefore, this study reveals that the divalent metal-dependent DNA cleavage activity is an intrinsic property of SOD1, which is independent of its natural metal sites.2. The roles of exogenous divalent metals in the nucleolytic activity were explored in detail by a series of biochemical experiments. Based on a non-equivalent multi-site binding model, affinity of a divalent metal for the enzyme-DNA complex was determined by absorption titration, indicating that the complex can provide at least a high and a low affinity site for the metal ion. These mean that the SOD1 may use a‘‘two exogenous metal ion pathway’’as a mechanism in which both metal ions are directly involved in the catalytic process of DNA cleavage. In addition, the pH versus DNA cleavage rate profiles can be fitted to two ionizing group models, indicating the presence of a general acid and a general base in catalysis. A model that requires histidine residues, metal bound water molecules and two hydrated metal ions to operate in concert could be used to interpret the catalysis of DNA hydrolysis, supported by the dependences of loss of the nucleolytic activity on time and on the concentration of the specific chemical modifier to the histidine residues on the enzyme.3. The aggregation behavior of SOD1 in the presence of DNA were examined under acidic conditions, which could mimic the effect of mutations and reflect the practical process done under physiological conditions to a high extent. Several forms of double-stranded DNA were tested to trigger the SOD1 aggregation by light scattering, single- and double-fluorescence imaging with the dyes, atomic force microscopy, and direct observations under visible light. The results reveal that DNA acts as a template for accelerating the formation of SOD1 aggregates and is incorporated into SOD1 aggregates. A significant alteration in hydrophobicity of SOD1 caused by both low pH and interactions with DNA, and the enrichment in SOD1 along DNA double strands are two main reasons responsible for DNA-accelerated SOD1 aggregation.4. The morphology of DNA-accelerated SOD1 aggregates was examined by transmission electron microscope, atomic force microscopy and fluorescence imaging of ThS. Several types of SOD1 aggregates were observed, which depend on the concentration of SOD1 and DNA, and the type of DNA molecules. DNA formed a compact structure in the high ratio of DNA to SOD1, and an incompact structure in the low ratio. The results reveal that both SOD1 aggregation and DNA condensation are coupled each other. Some DNA condensates could aggregate together to form fractal structures on mica and class, which followed the diffusion limited aggregation model.5. The acceleration effect of SOD1 aggregation in vitro upon addition of single-stranded DNA (ssDNA) of 24 nucleotides was examined under acidic conditions. ssDNA was tested to trigger the SOD1 aggregation by light scattering, single- and double-fluorescence imaging with the dyes, direct observations under visible light,atomic force microscopy and transmission electron microscope. The results reveal that ssDNA can accelerate the formation of ssDNA-SOD1 aggregate monomer, oligomeric aggregate, microaggregate, and macroaggregate. A significant alteration in hydrophobicity of SOD1 caused by both low pH and interactions with ssDNA, removal of the positive net charges of SOD1 by ssDNA, and the enrichment in SOD1 along ssDNA are driving forces for the rapid SOD1 aggregation. All these results indicate that the ssDNA-accelerated formation of insoluble SOD1 aggregates can act as a potential pathway to avoid accumulation of soluble SOD1 oligomeric intermediates. The small DNAs that are easily synthesized to target at protein oligomers might lower pathological consequences of SOD1.6. The acceleration effect of oxidized SOD1 aggregation in vitro upon addition of DNA was examined by gel electrophoresis, light scattering, and transmission electron microscope under physiological conditions. The results reveal that DNA can accelerate SOD1 aggregation when both SOD1 and DNA are in oxidative solution. A significant increase in hydrophobicity of SOD1 caused by oxidative modification, and the enrichment in SOD1 along DNA double strands are two main reasons responsible for the accelerated SOD1 aggregation. This result also implies that SOD1 of any one form with increased hydrophobicity, whatever caused by mutation or exogenous factor, may be accelerated to form aggregates due to the enrichment effect of DNA. This abnormal interaction with DNA may be a toxic gain of function of misfolded SOD1, which could cause ALS.更多还原