Jiang Wei, Han Yingchun, Zhou Ruoyu, Zhang Lina, Liu Changlin
Department of Chemistry, Huazhong University of Science and Technology, Wuhan 430074, China.
Biochemistry. 2007 May 22;46(20):5911-23. doi: 10.1021/bi062234m. Epub 2007 May 1.
The proteinaceous aggregates rich in copper, zinc superoxide dismutase (SOD1) have been shown to be involved in pathogenesis of amyotrophic lateral sclerosis (ALS). Since negatively charged species such as nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases, we examined here the aggregation behavior of SOD1 in the presence of DNA under acidic conditions that facilitate protein aggregation. Several forms of double-stranded DNA were tested to trigger SOD1 aggregation by light scattering, single- and double-fluorescence imaging with 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. The spherical and ellipsoidal SOD1 aggregates were characterized in both hydrated and dried states and have morphology similar to those identified in the diseased neurons. Light scattering experiments indicate that the aggregation first undergoes a rapid phase where the aggregates with average diameters of 40-80 nm rapidly form in <2 min, and then passes through a slow phase where the average diameters of aggregates were increased to at least 200-260 nm in 2 h. All forms of DNAs tested can lead to the aggregation of SOD1 at nanomolar levels. The association of SOD1 with DNA, driven by electrostatic interactions between both, can restrict the orientation of SOD1 molecules and increase a SOD1 population along DNA strands. This facilitates the hydrophobic interactions between SOD1 molecules, as indicated by hydrophobic probe binding and chemical denaturant treatment experiments. Demonstration of the DNA-accelerated aggregation of SOD1 might establish a possible role of DNA in the pathogenesis of some diseases because of the ubiquitous expression of SOD1 and the coexistence of SOD1 and DNA in the crowded molecular environment of a cell.
富含铜、锌超氧化物歧化酶（SOD1）的蛋白质聚集体已被证明与肌萎缩侧索硬化症（ALS）的发病机制有关。由于在淀粉样疾病患者的组织中经常发现带负电荷的物质如核酸与蛋白质沉积物相关联，我们在此研究了在促进蛋白质聚集的酸性条件下，SOD1在DNA存在时的聚集行为。通过光散射、用染料进行单荧光和双荧光成像、原子力显微镜以及在可见光下直接观察，测试了几种形式的双链DNA以触发SOD1聚集。结果表明，DNA作为模板加速SOD1聚集体的形成，并被纳入SOD1聚集体中。对球形和椭圆形的SOD1聚集体在水合和干燥状态下进行了表征，其形态与在患病神经元中鉴定出的形态相似。光散射实验表明，聚集首先经历一个快速阶段，在此阶段平均直径为40 - 80 nm的聚集体在不到2分钟内迅速形成，然后进入一个缓慢阶段，在此阶段聚集体的平均直径在2小时内增加到至少200 - 260 nm。所有测试的DNA形式都能在纳摩尔水平上导致SOD1聚集。SOD1与DNA的结合由两者之间的静电相互作用驱动，可限制SOD1分子的取向，并增加沿DNA链的SOD1数量。如疏水探针结合和化学变性剂处理实验所示，这促进了SOD1分子之间的疏水相互作用。由于SOD1的普遍表达以及在细胞拥挤的分子环境中SOD1和DNA的共存，证明DNA加速SOD1聚集可能确立了DNA在某些疾病发病机制中的可能作用。
