RESUMEN
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.
Asunto(s)
Ácidos Docosahexaenoicos/metabolismo , Peróxido de Hidrógeno/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ácidos Grasos/metabolismo , Humanos , Multimerización de Proteína , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Lipid peroxidation is a well-known process that has been implicated in many diseases. Recent evidence has shown that mitochondrial cholesterol levels are increased under specific conditions, making it an important target for peroxidation inside the mitochondria. Cholesterol peroxidation generates, as primary products, several hydroperoxides (ChOOH), which can react with transition metals and metalloproteins. In this sense, cytochrome c (CYTC), a heme protein largely found in the mitochondria, becomes a candidate to react with ChOOH. Using CYTC associated with SDS micelles to mimic mitochondrial conditions, we show that ChOOH induces dose-dependent CYTC Soret band bleaching, indicating that it is using ChOOH as a substrate. This reaction leads to protein oligomerization, suggesting the formation of a protein radical that, subsequently, recombines, giving dimers, trimers, and tetramers. EPR experiments confirmed the production of carbon-centered radicals from both protein and lipid in the presence of ChOOH. Similar results were obtained with linoleic acid hydroperoxides (LAOOH). In addition, replacing SDS micelles by cardiolipin-containing liposomes as the mitochondrial mimetic led to similar results with either ChOOH or LAOOH. Importantly, kinetic experiments show that CYTC bleaching is faster with ChOOH than with H2O2, suggesting that these hydroperoxides could be relevant substrates for CYTC peroxidase-like activity in biological media. Altogether, these results show that CYTC induces homolytic cleavage of lipid-derived hydroperoxides, producing lipid and protein radicals.
Asunto(s)
Colesterol/análogos & derivados , Citocromos c/química , Radicales Libres/química , Animales , Bovinos , Colesterol/química , Peróxido de Hidrógeno/química , Cinética , Ácidos Linoleicos/química , Peroxidación de Lípido , Peróxidos Lipídicos/química , Liposomas , Micelas , Polimerizacion , Piridinas/química , Dodecil Sulfato de SodioRESUMEN
Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid that is highly enriched in the brain, and the oxidation products of DHA are present or increased during neurodegenerative disease progression. The characterization of the oxidation products of DHA is critical to understanding the roles that these products play in the development of such diseases. In this study, we developed a sensitive and specific analytical tool for the detection and quantification of twelve major DHA hydroperoxide (HpDoHE) and hydroxide (HDoHE) isomers (isomers at positions 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 and 20) in biological systems. In this study, HpDoHE were synthesized by photooxidation, and the corresponding hydroxides were obtained by reduction with NaBH4. The isolated isomers were characterized by LC-MS/MS, and unique and specific fragment ions were chosen to construct a selected reaction monitoring (SRM) method for the targeted quantitative analysis of each HpDoHE and HDoHE isomer. The detection limits for the LC-MS/MS-SRM assay were 1-670 pg for HpDoHE and 0.5-8.5 pg for HDoHE injected onto a column. Using this method, it was possible to detect the basal levels of HDoHE isomers in both rat plasma and brain samples. Therefore, the developed LC-MS/MS-SRM can be used as an important tool to identify and quantify the hydro(pero)xy derivatives of DHA in biological system and may be helpful for the oxidative lipidomic studies.
Asunto(s)
Cromatografía Liquida/métodos , Ácidos Docosahexaenoicos/química , Hidróxidos/química , Peróxidos Lipídicos/química , Espectrometría de Masas en Tándem/métodos , Animales , Encéfalo , Isomerismo , Oxidación-Reducción , Ratas , Ratas Sprague-DawleyRESUMEN
Mitochondrial cholesterol has been reported to be increased under specific pathological conditions associated with enhanced oxidative stress parameters. In this scenario, cholesterol oxidation would be increased, leading to the production of reactive aldehydes, including cholesterol carboxyaldehyde (ChAld). By using SDS micelles as a mitochondrial mimetic model, we have demonstrated that ChAld covalently modifies cytochrome c (cytc), a protein known to participate in electron transport and apoptosis signaling. This mimetic model induces changes in cytc structure in the same way as mitochondrial membranes do. Tryptic digestion of the cytc-ChAld adduct followed by MALDI-TOF/TOF analyses revealed that modifications occur at Lys residues (K22) localized at cytc site L, a site involved in protein-protein and protein-membrane interactions. Interestingly, ChAld ligation prevented cytc detachment from liposomes even under high ionic strength conditions. Overall, it can be concluded that ChAld ligation to Lys residues at site L creates a hydrophobic tail at cytc, which promotes cytc anchoring to the membrane. Although not investigated in detail in this study, cytc adduction to cholesterol derived aldehydes could have implications in cytc release from mitochondria under apoptotic stimuli.
Asunto(s)
Aldehídos/química , Materiales Biomiméticos/metabolismo , Colesterol/análogos & derivados , Colesterol/química , Citocromos c/metabolismo , Liposomas/metabolismo , Mitocondrias/metabolismo , Aldehídos/toxicidad , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Materiales Biomiméticos/química , Bovinos , Citocromos c/química , Transporte de Electrón/efectos de los fármacos , Luz , Liposomas/química , Lisina/química , Modelos Moleculares , Miocardio/metabolismo , Oxidación-Reducción , Péptidos/análisis , Péptidos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Tripsina/metabolismoRESUMEN
Cardiolipin, the main anionic phospholipid in the inner mitochondrial membrane, provides shape, charge and osmotic support to this membrane due to its biophysical properties. In addition, it helps form respiratory supercomplexes and provides functionality to mitochondrial proteins. Defects in the biosynthesis or remodeling of cardiolipin have been related to severe diseases, such as Barth syndrome. Opi1p, a transcriptional repressor for most enzymes in phospholipid biosynthesis found in Saccharomyces cerevisiae, has been demonstrated not to affect the biosynthesis of this mitochondrial phospholipid. However, we found that opi1 deletion compromises mitochondrial metabolism producing severe respiratory defects. The mechanism producing this phenotype was explored and found to be a mitochondrial cardiolipin depletion of almost 50%, resulting in low cytochrome content and high mitochondrial DNA instability. The origin of this low cardiolipin content strongly correlated with the overproduction of inositol, an intrinsic phenotype of this mutation. Overall, our results show that adequate regulation of phospholipid synthesis is essential for the maintenance of mitochondrial function.
Asunto(s)
Cardiolipinas/metabolismo , Mitocondrias/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cardiolipinas/biosíntesis , ADN de Hongos/genética , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/biosíntesis , Dosificación de Gen/genética , Inestabilidad Genómica/genética , Inositol/biosíntesis , Inositol/metabolismo , Membranas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/biosíntesis , ATPasas de Translocación de Protón/biosíntesis , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
A Esclerose Lateral Amiotrófica (ELA) é uma doença progressiva e fatal causada pela degeneração seletiva dos neurônios motores do cérebro e medula. Dos casos familiares de ELA (fELA), 20% são causados por mutações pontuais no gene da sod1. O ácido docosahexaenoico (C22:6, n-3, DHA) é um ácido graxo altamente insaturado, sendo um dos principais ácidos graxos da massa cinzenta do cérebro. Estudos têm correlacionado mutações de SOD1 com a formação de agregados que poderiam ser induzidos por ácidos graxos insaturados. O objetivo deste estudo foi avaliar os efeitos e mecanismos do DHA e de seus hidroperóxidos (DHAOOH) na agregação de SOD1 in vitro. As análises de dicroísmo circular (CD) mostraram mudanças na estrutura secundária de ambas as proteínas apo-SOD1WT e G93A promovidas pelo DHA, resultando em aumento de superfície hidrofóbica e formação de estruturas do tipo beta-amilóide, como mostrado pelos ensaios do bis- ANS e Tioflavina, respectivamente. Estas mudanças resultam na formação de agregados amorfos como observado por microscopia eletrônica de varredura (MEV). Espécies de alto peso molecular foram observadas nas incubações do DHA com as formas apo da SOD1 por SDS-PAGE sob condições não redutoras e também por cromatografia de exclusão por tamanho. A formação dos agregados mostrou-se dependente de resíduos de Cys na sua forma desprotonada, visto que agregados não foram observados na presença de beta-mercaptoetanol e sua formação foi inibida na presença de bloqueador de tióis e em pH ácido. Além disso, análises por cromatografia de exclusão mostraram que a agregação é dependente da insaturação e conformação cis dos ácidos graxos. Comparativamente ao DHA, os hidroperóxidos do DHA tiveram um efeito menor na agregação de SOD1, porém revelaram a propriedade de induzir a dimerização covalente de SOD1. No geral, os dados mostram que o DHA induz a agregação de SOD1, através de um processo envolvendo a exposição de superfícies hidrofóbicas, formação de pontes dissulfeto e também de possíveis cross-links envolvendo reações do tipo "ene-tiol"
ALS is a progressive and fatal disease caused by selective degeneration of motor neurons in the brain and spinal cord. Twenty percent of familial ALS (fALS) cases are caused mainly by point mutations in the sod1 gene. Docosahexaenoic acid (C22:6, n-3, DHA) is a highly unsaturated fatty acid, wich is one of the main fatty acids in the cerebral gray matter. Studies have linked SOD1 mutations to the formation of aggregates that could be induced by unsaturated fatty acids. The aim of this study was to evaluate the effect of DHA on aggregation of SOD1 fALS mutants in vitro and its mechanisms. CD analysis shows changes in the secondary structure of both apo-SOD1WT and G93A promoted by DHA resulting in an increase in the surface hydrophobicity and formation of structures such as beta amyloid, which was also confirmed by bis-ANS assay and Thioflavin, respectively. These changes enhance the interaction of SOD1 and DHA, leading to amorphous aggregates as revealed by FESEM. Incubation of DHA with apo-SOD1 forms results in high-molecular weight species as detected by SDS-PAGE analyses under non-reducing conditions and also by size exclusion chromatography. This appears to require Cys residues in their thiolate forms because high aggregates are not observed under reducing conditions and also by size exclusion chromatography or at acidic pH. Also, size-exclusion chromatography indicates that the mutant apo-SOD1 aggregation is dependent on the unsaturation and cis-conformation of fatty acids. Compared to the DHA, DHAOOH had a minor effect on SOD1 aggregation, however revealed the ability to induce covalent dimerization of SOD1. Overall, the data suggest a mechanism of DHA aggregation, by a process involving exposure to hydrophobic surfaces, formation of disulfide bonds and also for possible cross-links involving reactions such "thiol-ene"