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首页> 外文学位 >Copper-zinc superoxide dismutase and amyotrophic lateral sclerosis: Solution dynamics of the apoproteins and shotgun proteomics of SOD1 aggregates purified from transgenic mouse.
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Copper-zinc superoxide dismutase and amyotrophic lateral sclerosis: Solution dynamics of the apoproteins and shotgun proteomics of SOD1 aggregates purified from transgenic mouse.

机译：铜锌超氧化物歧化酶和肌萎缩性侧索硬化：从转基因小鼠中纯化的SOD1聚集体的脱辅基蛋白和shot弹枪蛋白质组学的溶液动力学。

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摘要

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that involves the selective death of motor neurons. Nearly 2% of ALS cases are caused by mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). These SOD1 mutations cause ALS by imparting a toxic function to the protein, in addition to its normal, beneficial function as a superoxide anion scavenger. An abundance of experimental evidence points towards SOD1 aggregation as the underlying cause of SOD1 neurotoxicity and it is thought that pathogenic mutations to SOD1 increase the aggregation propensity of the SOD1 polypeptide. This thesis reports hydrogen-deuterium (H/D) exchange and differential scanning calorimetry (DSC) data that demonstrates ALS mutations to SOD1 do not uniformly destabilize the native fold of the polypeptide. Moreover, many ALS mutations do not perturb any studied property of SOD1 such as metal coordination or SOD activity. Hence, destabilization of the SOD1 native state is insufficient to completely explain the neurodegenerative effects of ALS mutations in SOD1. Additionally, this thesis presents site-specific H/D exchange data on the metal free (apo) form of the A4V variant of SOD1. The A4V mutation is the most frequently occurring ALS-linked mutation to SODI. H/D exchange monitored by mass spectrometry revealed that many regions of the folded SOD1 polypeptide were not perturbed by the A4V substitution. However, one region of the protein, residues 50-53, of the disulfide sub-loop and dimer interface, exhibited no protection from H/D exchange in the A4V apoprotein and this was in contrast to the wild-type (WT) SOD1 protein. A subpopulation of A4V apo-SOD1 was also observed to undergo slow, localized unfolding at residues 21-53, which represents beta-strands 3 and 4. Upon reduction of the intramolecular disulfide bond, A4V apo-SOD1 exhibited no detectable endothermic transition with DSC and exchanged with D2O rapidly, at a rate characteristic of a random coil polypeptide. This thesis also describes a sequential size exclusion/immunoaffinity chromatography method for purifying SOD1 and SOD1 aggregates from the spinal cord of transgenic mice carrying the human SOD1 gene with various ALS mutations. A mass spectrometry based proteomic analysis of purified samples revealed that calmodulin-3, glial fibrillary acidic protein, neurofilament, and various membrane proteins from the mitochondrial electron transport chain were present with mutant hSOD1 in high molecular weight (>70 kDa) complexes. Additionally, MALDI-TOF MS showed that various mutant SOD1 proteins derived from these high molecular weight complexes also possessed apparent low molecular weight modifications (100 Da). In lower molecular weight complexes (70 kDa), mutant SOD1 was observed to be associating with Bcl-2 associated protein-X (BAX).
机译：肌萎缩性侧索硬化症（ALS）是一种致命的神经退行性疾病，涉及运动神经元的选择性死亡。几乎2％的ALS病例是由编码铜锌超氧化物歧化酶（SOD1）的基因突变引起的。这些SOD1突变除了赋予蛋白质作为超氧化物阴离子清除剂的正常有益功能外，还通过赋予蛋白质毒性功能而导致ALS。大量的实验证据表明SOD1聚集是SOD1神经毒性的根本原因，人们认为SOD1的致病性突变会增加SOD1多肽的聚集倾向。本论文报道了氢-氘（H / D）交换和差示扫描量热法（DSC）数据，这些数据证明ALS突变为SOD1不会均匀地破坏多肽的天然折叠。此外，许多ALS突变不会干扰SOD1的任何研究性质，例如金属配位或SOD活性。因此，SOD1原始状态的不稳定不足以完全解释SOD1中ALS突变的神经退行性作用。此外，本文提出了SOD1 A4V变体的无金属（apo）形式的特定位置H / D交换数据。 A4V突变是最常见的与SODI关联的ALS突变。通过质谱监测的H / D交换显示，折叠的SOD1多肽的许多区域不受A4V取代的干扰。然而，该蛋白的一个区域，即二硫键亚环和二聚体界面的残基50-53，在A4V脱辅基蛋白中未显示出不受H / D交换的保护作用，这与野生型（WT）SOD1蛋白相反。还观察到A4V apo-SOD1的一个亚群在代表β链3和4的残基21-53处经历缓慢的局部展开。分子内二硫键还原后，A4V apo-SOD1表现出未检测到DSC的吸热转变并以无规卷曲多肽特征的速率迅速与D2O交换。本论文还描述了一种顺序大小排阻/免疫亲和层析方法，该方法用于从携带带有各种ALS突变的人SOD1基因的转基因小鼠的脊髓中纯化SOD1和SOD1聚集体。基于质谱的蛋白质组学分析表明，钙调蛋白-3，胶质纤维酸性蛋白，神经丝和来自线粒体电子传输链的各种膜蛋白与突变型hSOD1呈高分子量（> 70 kDa）的复合物。此外，MALDI-TOF MS表明，衍生自这些高分子量复合物的各种突变型SOD1蛋白也具有明显的低分子量修饰（<100 Da）。在较低分子量的复合物中（<70 kDa），观察到突变型SOD1与Bcl-2相关蛋白X（BAX）相关。

著录项

  • 作者

    Shaw, Bryan Francis.;

  • 作者单位

    University of California, Los Angeles.;

  • 授予单位 University of California, Los Angeles.;
  • 学科 Biology Neuroscience.; Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 161 p.
  • 总页数 161
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 神经科学;生物化学;
  • 关键词

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