Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons leading to muscle paralysis and death. While a link between dysregulated lipid metabolism and ...ALS has been proposed, lipidome alterations involved in disease progression are still understudied. Using a rodent model of ALS overexpressing mutant human Cu/Zn-superoxide dismutase gene (SOD1-G93A), we performed a comparative lipidomic analysis in motor cortex and spinal cord tissues of SOD1-G93A and WT rats at asymptomatic (~70 days) and symptomatic stages (~120 days). Interestingly, lipidome alterations in motor cortex were mostly related to age than ALS. In contrast, drastic changes were observed in spinal cord of SOD1-G93A 120d group, including decreased levels of cardiolipin and a 6-fold increase in several cholesteryl esters linked to polyunsaturated fatty acids. Consistent with previous studies, our findings suggest abnormal mitochondria in motor neurons and lipid droplets accumulation in aberrant astrocytes. Although the mechanism leading to cholesteryl esters accumulation remains to be established, we postulate a hypothetical model based on neuroprotection of polyunsaturated fatty acids into lipid droplets in response to increased oxidative stress. Implicated in the pathology of other neurodegenerative diseases, cholesteryl esters appear as attractive targets for further investigations.
Cytochrome c (cytc) is a heme protein of 12 kDa that transfers electrons in the mitochondrial respiratory chain. Increased cytc peroxidase activity leads to cardiolipin (CL) oxidation, a hallmark of ...early apoptosis stage. Here, we aimed to investigate the interaction between cytc with cardiolipin hydroperoxide (CLOOH) in a mimetic mitochondrial membrane. Cytc-CL peroxidase reaction occurred at faster rates with CLOOH than with H2O2. Moreover, liposomes containing CLOOH promoted increased protein aggregation with minor or no release of cytc from the membrane. Dimeric and trimeric cytc species were observed in the first 15 min, followed by increased formation of high-molecular-weight aggregates afterwards. nLC-MS/MS analysis identified several Lys and His residues covalently modified by lipid aldehydes that showed mass increments corresponding to 4-hydroxynonenal (HNE), 4-oxononenal (ONE), hexanoyl, heptenal and octenal addition. Noteworthy, most modifications were observed at Lys and His residues located at A-site (K73, K87, K88), L-site (H26, H33, and K27) membrane binding sites. Further, dityrosine cross-linked peptides were also characterized at residues Y48-Y74, Y48-Y97 and Y74-Y97. Collectively, our findings show that CLOOH causes irreversible protein damage and crosslinking of cytc in the membrane.
•Cytc reacts with cardiolipin hydroperoxides at faster rates than with hydrogen peroxide.•Cytc binds to liposomes containing cardiolipin hydroperoxides and becomes highly aggregated.•Lys and His residues involved in cytc-CL electrostatic binding sites are modified by aldehydes.•Tyr residues located at cytc surface are involved in dityrosine crosslinking.
Cardiolipin is the signature phospholipid of the mitochondrial inner membrane. It participates in shaping the inner membrane as well as in modulating the activity of many membrane-bound proteins. The ...acyl chain composition of cardiolipin is finely tuned post-biosynthesis depending on the surrounding phospholipids to produce mature or unsaturated cardiolipin. However, experimental evidence showing that immature and mature cardiolipin are functionally equivalents for mitochondria poses doubts on the relevance of cardiolipin remodeling. In this work, we studied the role of cardiolipin acyl chain composition in mitochondrial bioenergetics, including a detailed bioenergetic profile of yeast mitochondria. Cardiolipin acyl chains were modified by genetic and nutritional manipulation. We found that both the bioenergetic efficiency and osmotic stability of mitochondria are dependent on the unsaturation level of cardiolipin acyl chains. It is proposed that cardiolipin remodeling and, consequently, mature cardiolipins play an important role in mitochondrial inner membrane integrity and functionality.
•External linoleic acid is incorporated into phospholipids.•Polyunsaturated cardiolipin improves the oxidative capacity of mitochondria.•Mitochondrial osmotic stability enhances when mitochondria is enriched in unsaturated cardiolipins.•A high proportion of saturated cardiolipins in mitochondria led to osmotic instability.
Cytochrome c (cyt c) is a heme protein of 12 kDa that transfers electrons in the mitochondrial respiratory chain. Several in vitro studies have shown that increased cyt c peroxidase activity leads to ...cardiolipin (CL) oxidation, a hallmark of early stages of apoptosis. Apart from CL hydroperoxide (CL-OOH) and hydroxide (CL-OH) formation, it is also known that cyt c aggregates when interacting with H2O2. Protein aggregation can play an important role in cell signaling, but a definitive characterization remains to be performed. Here we aimed to investigate the role of CL-OOH in cyt c post-translational modifications and aggregation. Analysis of free cyt c after ultracentrifugation showed that cyt c remains anchored to liposomes (composed of phosphatidylcholine and CL (80:20 mol/mol) containing 20% and 100% of the CL as CL-OOH. This was confirmed using a high-ionic strength KCl-based medium, leading to the hypothesis that CL-OOH may induce covalent binding of cyt c to the membrane. This binding was further demonstrated to be time-dependent, with dimeric and trimeric species observed in the first 15 min and increased aggregates formation afterwards. We estimated this reaction rate at 9.58 ± 0.16 x 102k x M-1 x s-1, which is 100 times faster than that with H2O2. Data on nano-liquid chromatography coupled to mass spectrometry (Q-TOF) have revealed both ONE and HNE modification sites on lysine (K27, K73 and K88) and arginine residues as well as dityrosine cross-linking between Y74 and Y94 residues. These cross-linking pathways may play a role in cyt c oligomerization. Our findings thus suggest that CL-OOH can induce cyt c covalent binding to membranes and protein cross-linking. Furthermore, formation of K27, K73 and K88 adducts by CL-OOH derived electrophiles represents potential sites for lipid-protein interaction. Collectively, our results may shed light into the role of CL-OOH in cell death signaling in biological systems.
Acknowledgements
FAPESP, CNPq, CAPES, INCT/NAP/CEPID Redoxoma.
A Síndrome Doença Celíaca, Epilepsia e Calcificação Cerebral (CEC) é uma patologia rara e pouco conhecida que pode ser confundida com outras condições neurológicas. Os exames de imagem são ...fundamentais para o diagnóstico correto e preciso, os quais permitem a escolha do tratamento mais adequado. O objetivo deste trabalho é relatar um caso de síndrome CEC em um paciente com diagnóstico prévio de doença celíaca e epilepsia, ao destacar a importância dos exames de imagem no diagnóstico e no tratamento dessa patologia. Um paciente de 29 anos, sexo masculino, apresentou quadro de migrânea crônica e amaurose há 5 anos, além de histórico de doença celíaca (DC) e epilepsia. O paciente foi admitido no centro de neurorradiologia de um hospital de referência na capital paulista para investigação diagnóstica. Foram realizados exames de imagens: ressonância magnética (RM) do crânio e tomografia computadorizada (TC). A RM evidenciou extensos focos de hipossinal associados a áreas de encefalomalácia e gliose, os quais acometeram a substância branca dos lobos occipitais, com extensão aos lobos temporais e parietais. A TC evidenciou extensas calcificações grosseiras que acometeram as regiões parieto-temporo-occipitais, associadas a uma área hipoatenuante no parênquima encefálico adjacente sugestivo de gliose. Frente à análise dos resultados dos exames de imagens realizados, confirmou-se o diagnóstico de síndrome CEC, tendo em vista o diagnóstico prévio de DC e a associação com o histórico de crises convulsivas e os achados imagenológicos relativos às calcificações subcorticais occipitais, bilaterais e simétricas presentes, além da ausência de efeito expansivo e realce pelo meio de contraste. A síndrome CEC é uma patologia rara e pouco conhecida que pode apresentar sintomas semelhantes a outras doenças neurológicas. O diagnóstico correto e preciso é essencial para o tratamento adequado e para a prevenção de complicações futuras. Os exames de imagem, como a ressonância magnética e a tomografia computadorizada, são fundamentais para o diagnóstico e a escolha do tratamento mais adequado. No caso descrito, o uso do levetiracetam foi eficaz para o controle das crises epilépticas e para a melhora da qualidade de vida do paciente. É importante que os profissionais de saúde estejam atentos aos sintomas e ao histórico do paciente para um diagnóstico precoce e preciso da patologia.
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