Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Mol Neurodegener. 2013 Nov 21;8:43. doi: 10.1186/1750-1326-8-43.
Mutations in the Cu/Zn superoxide dismutase gene (SOD1) are responsible for 20% of familial forms of amyotrophic lateral sclerosis (ALS), and mutant SOD1 has been shown to have increased surface hydrophobicity in vitro. Mutant SOD1 may adopt a complex array of conformations with varying toxicity in vivo. We have used a novel fluorescence-based proteomic assay using 4,4'-bis-1-anilinonaphthalene-8-sulfonate (bisANS) to assess the surface hydrophobicity, and thereby distinguish between different conformations, of SOD1 and other proteins in situ.
Covalent bisANS labeling of spinal cord extracts revealed that alterations in surface hydrophobicity of H46R/H48Q mutations in SOD1 provoke formation of high molecular weight SOD1 species with lowered solubility, likely due to increased exposure of hydrophobic surfaces. BisANS was docked on the H46R/H48Q SOD1 structure at the disordered copper binding and electrostatic loops of mutant SOD1, but not non-mutant WT SOD1. 16 non-SOD1 proteins were also identified that exhibited altered surface hydrophobicity in the H46R/H48Q mutant mouse model of ALS, including proteins involved in energy metabolism, cytoskeleton, signaling, and protein quality control. Heat shock proteins (HSPs) were also enriched in the detergent-insoluble fractions with SOD1. Given that chaperones recognize proteins with exposed hydrophobic surfaces as substrates and the importance of protein homeostasis in ALS, we crossed SOD1 H46R/H48Q mutant mice with mice over-expressing the heat shock factor 1 (HSF1) transcription factor. Here we showed that HSF1 over-expression in H46R/H48Q ALS mice enhanced proteostasis as evidenced by increased expression of HSPs in motor neurons and astrocytes and increased solubility of mutant SOD1. HSF1 over-expression significantly reduced body weight loss, delayed ALS disease onset, decreases cases of early disease, and increased survival for the 25th percentile in an H46R/H48Q SOD1 background. HSF1 overexpression did not affect macroautophagy in the ALS background, but was associated with maintenance of carboxyl terminus of Hsp70 interacting protein (CHIP) expression which declined in H46R/H48Q mice.
Our results uncover the potential importance of changes in protein surface hydrophobicity of SOD1 and other non-SOD1 proteins in ALS, and how strategies that activate HSF1 are valid therapies for ALS and other age-associated proteinopathies.
铜锌超氧化物歧化酶基因（SOD1）的突变导致 20%的家族性肌萎缩侧索硬化症（ALS），体外研究表明突变型 SOD1 的表面疏水性增加。突变型 SOD1 可能在体内采用一系列具有不同毒性的复杂构象。我们使用了一种新型基于荧光的蛋白质组学测定方法，使用 4,4'-双-1-苯胺基萘-8-磺酸钠（bisANS）来评估 SOD1 和其他蛋白质的表面疏水性，从而区分其不同构象。
共价 bisANS 标记脊髓提取物表明，H46R/H48Q 突变 SOD1 的表面疏水性改变导致高分子量 SOD1 物种的形成，其溶解度降低，可能是由于疏水面的暴露增加。BisANS 被对接在 H46R/H48Q SOD1 结构上，位于突变 SOD1 的无序铜结合和静电环上，但不在非突变 WT SOD1 上。在 ALS 的 H46R/H48Q 突变小鼠模型中，还鉴定出 16 种非 SOD1 蛋白，其表面疏水性发生改变，包括参与能量代谢、细胞骨架、信号转导和蛋白质质量控制的蛋白质。热休克蛋白（HSPs）也在去污剂不溶性部分中富集，与 SOD1 一起。鉴于伴侣蛋白将暴露的疏水面作为底物识别蛋白质，以及蛋白质平衡在 ALS 中的重要性，我们将 SOD1 H46R/H48Q 突变小鼠与过表达热休克因子 1（HSF1）转录因子的小鼠进行了杂交。在这里，我们表明 HSF1 在 H46R/H48Q ALS 小鼠中的过表达增强了蛋白质平衡，这表现为运动神经元和星形胶质细胞中 HSPs 的表达增加，以及突变 SOD1 的溶解度增加。HSF1 的过表达显著减少了体重减轻，延迟了 ALS 疾病的发作，减少了早期疾病的病例，并增加了 H46R/H48Q SOD1 背景下第 25 百分位的存活时间。在 ALS 背景下，HSF1 的过表达并不影响巨自噬，但与羧基末端热休克蛋白 70 相互作用蛋白（CHIP）表达的维持有关，而 H46R/H48Q 小鼠中的 CHIP 表达下降。
我们的结果揭示了 SOD1 和其他非 SOD1 蛋白质表面疏水性变化在 ALS 中的潜在重要性，以及激活 HSF1 的策略如何成为 ALS 和其他与年龄相关的蛋白质病的有效治疗方法。
