Oligomerization of Cu,Zn-Superoxide Dismutase (SOD1) by Docosahexaenoic Acid and Its Hydroperoxides In Vitro: Aggregation Dependence on Fatty Acid Unsaturation and Thiols.
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.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Patricia Postilione
PP
Appolinário
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Danilo Bilches
DB
Medinas
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Adriano B
AB
Chaves-Filho
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Thiago C
TC
Genaro-Mattos
Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
José Renato Rosa
JR
Cussiol
Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
Luis Eduardo Soares
LE
Netto
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Ohara
O
Augusto
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Sayuri
S
Miyamoto
D002850:Chromatography, Gel; D004281:Docosahexaenoic Acids / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D055503:Protein Multimerization; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism*
D005227:Fatty Acids; D013438:Sulfhydryl Compounds; D004281:Docosahexaenoic Acids; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase