Structural similarity of wild-type and ALS-mutant superoxide dismutase-1 fibrils using limited proteolysis and atomic force microscopy
Structural similarity of wild-type and ALS-mutant superoxide dismutase-1 fibrils using limited proteolysis and atomic force microscopy
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DOI：
10.1073/pnas.1309613110
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发表时间：
2013-07-02
影响因子：
11.1
通讯作者：
Valentine, Joan Selverstone
中科院分区：
文献类型：
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作者：
Chan, Pik K.;Chattopadhyay, Madhuri;Valentine, Joan Selverstone
由错误折叠的超氧化物歧化酶-1（SOD 1）蛋白形成的异常组装可能是SOD 1相关家族性肌萎缩侧索硬化症（fALS）的原因，并可能参与某些散发性ALS病例。为了分析不溶性SOD 1淀粉样纤维的结构，我们首先使用有限的蛋白酶解，然后进行质谱分析。用胰蛋白酶、糜蛋白酶或链霉蛋白酶消化由全长N-乙酰化WT SOD 1形成的淀粉样蛋白原纤维，显示N末端的前63个残基通过原纤维形成而免受蛋白酶消化。此外，每种测试的ALS突变体SOD 1蛋白（G37 R、L38 V、G41 D、G93 A、G93 S和D101 N）在胰蛋白酶消化后显示出类似的保护片段。我们的第二种结构表征方法使用原子力显微镜对SOD 1原纤维进行成像，并显示WT和突变体显示出相似的扭曲形态。WT原纤维具有62.1 nm的一致平均螺距距离。ALS突变体SOD 1蛋白L38 V、G93 A和G93 S形成具有与WT非常相似的螺旋扭曲模式的原纤维，而突变体蛋白G37 R、G41 D和D101 N观察到小但显著的结构偏差。总体而言，我们的研究表明，测试的人WT SOD 1和ALS突变体具有通过N末端进行置换的共同内在倾向，并且单个氨基酸取代可导致螺旋扭曲模式的变化。
Abnormal assemblies formed by misfolded superoxide dismutase-1 (SOD1) proteins are the likely cause of SOD1-linked familial amyotrophic lateral sclerosis (fALS) and may be involved in some cases of sporadic ALS. To analyze the structure of the insoluble SOD1 amyloid fibrils, we first used limited proteolysis followed by mass spectrometric analysis. Digestion of amyloid fibrils formed from full-length N-acetylated WT SOD1 with trypsin, chymotrypsin, or Pronase revealed that the first 63 residues of the N terminus were protected from protease digestion by fibril formation. Furthermore, every tested ALS-mutant SOD1 protein (G37R, L38V, G41D, G93A, G93S, and D101N) showed a similar protected fragment after trypsin digestion. Our second approach to structural characterization used atomic force microscopy to image the SOD1 fibrils and revealed that WT and mutants showed similar twisted morphologies. WT fibrils had a consistent average helical pitch distance of 62.1 nm. The ALS-mutant SOD1 proteins L38V, G93A, and G93S formed fibrils with helical twist patterns very similar to those of WT, whereas small but significant structural deviations were observed for the mutant proteins G37R, G41D, and D101N. Overall, our studies suggest that human WT SOD1 and ALS-mutants tested have a common intrinsic propensity to fibrillate through the N terminus and that single amino acid substitutions can lead to changes in the helical twist pattern.