Appolinário Patricia Postilione, Medinas Danilo Bilches, Chaves-Filho Adriano B, Genaro-Mattos Thiago C, Cussiol José Renato Rosa, Netto Luis Eduardo Soares, Augusto Ohara, Miyamoto Sayuri
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
PLoS One. 2015 Apr 30;10(4):e0125146. doi: 10.1371/journal.pone.0125146. eCollection 2015.
Docosahexaenoic acid (C22:6, n-3, DHA) is a polyunsaturated fatty acid highly enriched in the brain. This fatty acid can be easily oxidized yielding hydroperoxides as primary products. Cu, Zn-Superoxide dismutase (SOD1) aggregation is a common hallmark of Amyotrophic Lateral Sclerosis (ALS) and the molecular mechanisms behind their formation are not completely understood. Here we investigated the effect of DHA and its hydroperoxides (DHAOOH) on human SOD1 oligomerization in vitro. DHA induced the formation of high-molecular-weight (HMW) SOD1 species (>700 kDa). Aggregation was dependent on free thiols and occurred primarily with the protein in its apo-form. SOD1 incubation with DHA was accompanied by changes in protein structure leading to exposure of protein hydrophobic patches and formation of non-amyloid aggregates. Site-directed mutagenesis studies demonstrated that Cys 6 and Cys 111 in wild-type and Cys 6 in ALS-linked G93A mutant are required for aggregation. In contrast, DHAOOH did not induce HMW species formation but promoted abnormal covalent dimerization of apo-SOD1 that was resistant to SDS and thiol reductants. Overall, our data demonstrate that DHA and DHAOOH induce distinct types of apo-SOD1 oligomerization leading to the formation of HMW and low-molecular-weight species, respectively.
二十二碳六烯酸（C22:6，n-3，DHA）是一种在大脑中高度富集的多不饱和脂肪酸。这种脂肪酸很容易被氧化，产生氢过氧化物作为主要产物。铜锌超氧化物歧化酶（SOD1）聚集是肌萎缩侧索硬化症（ALS）的一个常见标志，其形成背后的分子机制尚未完全了解。在这里，我们研究了DHA及其氢过氧化物（DHAOOH）对人SOD1体外寡聚化的影响。DHA诱导形成高分子量（HMW）SOD1物种（>700 kDa）。聚集依赖于游离巯基，主要发生在脱辅基形式的蛋白质上。SOD1与DHA孵育伴随着蛋白质结构的变化，导致蛋白质疏水区域暴露和非淀粉样聚集体的形成。定点诱变研究表明，野生型中的Cys 6和Cys 111以及ALS相关G93A突变体中的Cys 6是聚集所必需的。相比之下，DHAOOH没有诱导HMW物种的形成，但促进了脱辅基SOD1的异常共价二聚化，这种二聚化对SDS和巯基还原剂具有抗性。总体而言，我们的数据表明，DHA和DHAOOH分别诱导不同类型的脱辅基SOD1寡聚化，导致形成HMW和低分子量物种。
