Cytochrome c reacts with cholesterol hydroperoxides to produce lipid- and protein-derived radicals.

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.

The development of a specific and sensitive LC-MS-based method for the detection and quantification of hydroperoxy- and hydroxydocosahexaenoic acids as a tool for lipidomic analysis.

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.

Covalent binding and anchoring of cytochrome c to mitochondrial mimetic membranes promoted by cholesterol carboxyaldehyde.

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.