cytochrome-c-t and methionine-sulfoxide

Met80 of cytochrome c (cyt c) has been shown to dissociate from its heme iron when cyt c interacts with cardiolipin (CL), which triggers the release of cyt c into the cytosol initiating apoptosis. We found that the mass of human cyt c increases by 16 Da in the Met80-Lys86 region by reaction with molecular oxygen in the presence of CL-containing liposomes and dithiothreitol (DTT). To investigate the effect of Met80 dissociation on the reaction of cyt c with molecular oxygen without affecting its secondary structures, a human cyt c mutant (Δ8384 cyt c) was constructed by removing two amino acids (Val83 and Gly84) from the loop containing Met80. According to MALDI-TOF-MS and tandem mass measurements, Met80 of Δ8384 cyt c was modified site-specifically to methionine sulfoxide when purified in the presence of molecular oxygen, whereas Met80 was not modified in the absence of molecular oxygen. A red-shift of the Soret band from 406 to 412 nm and absorption increase at ∼536 and ∼568 nm were observed for Δ8384 cyt c when it reacted with DTT and molecular oxygen, followed by a further red-shift of the Soret band to 416 nm and absorption increase at ∼620 and ∼650 nm. These results indicate that Met80 of cyt c is oxidized site-specifically by formation of the oxy and subsequent compound I-like species when Met80 dissociates from the heme iron, where the Met80 modification may affect its peroxidase activity related to apoptosis.

Topics: Cytochromes c; Dithiothreitol; Heme; Humans; Iron; Liposomes; Methionine; Oxidation-Reduction; Oxygen; Protein Conformation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Alzheimer disease (AD) affects mainly people over the age of 65 years, suffering from different clinical symptoms such as progressive decline in memory, thinking, language, and learning capacity. The toxic role of β-amyloid peptide (Aβ) has now shifted from insoluble Aβ fibrils to smaller, soluble oligomeric Aβ aggregates. The urgent need for efficient new therapies is high; robust models dissecting the physiopathological aspects of the disease are needed. We present here a model allowing study of four cytopathic effects of Aβ oligomers (AβO): oxidative stress, loss of synapses, disorganization of the neurite network, and cellular death. By generating a solution of AβO and playing on the concentration of and time of exposure to AβO, we have shown that it was possible to reproduce early effects (oxidative stress) and the long-term development of structural alterations (death of neurons). We have shown that 1) all toxic events were linked to AβO according to a specific timing and pathway and 2) AβO were probably the key intermediates in AD pathogenesis. The present model, using Aβ peptide solution containing AβO, reproduced essential neuropathological features of AD; the effects involved were similar whatever the kind of neurons tested (cortical vs. hippocampal). By using a single system, it was possible to embrace all toxic mechanisms at defined times and concentrations, to study each involved pathway, and to study the effects of new molecules on the different neurotoxic pathways responsible for development of AD.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Catalase; Cells, Cultured; Cerebral Cortex; Cytochromes c; Embryo, Mammalian; Female; Hippocampus; Methionine; Nerve Net; Neurons; Organ Culture Techniques; Oxidative Stress; Peptide Fragments; Pregnancy; Rats; Rats, Wistar; Synapses

Considerable evidence indicates a role for methionine sulfoxide reductase A (MsrA) in lens cell resistance to oxidative stress through its maintenance of mitochondrial function. Correspondingly, increased protein methionine sulfoxide (PMSO) is associated with lens aging and human cataract formation, suggesting that loss of MsrA activity is associated with this disease. Here we tested the hypothesis that loss of MsrA protein repair is associated with cataract formation. To test this hypothesis we examined the effect of MsrA deletion on lens opacity in mice treated with hyperbaric oxygen, identified lens mitochondrial proteins oxidized upon deletion of MsrA and determined the ability of MsrA to repair the identified proteins.. Wild-type and MsrA knockout mice were treated or not treated with 100 treatments of hyperbaric oxygen (HBO) over an 8 month period and lenses were examined by in vivo light scattering measurements documented by slit-lamp imaging. Co-immunoprecipitation of MsrA was conducted against five specific protein representatives of the five complexes of the electron transport chain in addition to cytochrome c (cyt c). Cyt c in lens protein from the knockout and wild-type lenses was subjected to cyanogen bromide (CNBr) cleavage to identify oxidized methionines. Methionine-specific CNBr cleavage was used to differentiate oxidized and un-oxidized methionines in cyt c in vitro and the ability of MsrA to restore the activity of oxidized cyt c was evaluated. Mass spectrometry analysis of cyt c was used to confirm oxidation and repair by MsrA in vitro.. HBO treatment of MsrA knockout mice led to increased light scattering in the lens relative to wild-type mice. MsrA interacted with four of the five complexes of the mitochondrial electron transport chain as well as with cyt c. Cyt c was found to be aggregated and degraded in the knockout lenses consistent with its oxidation. In vitro analysis of oxidized cyt c revealed the presence of two oxidized methionines (met 65 and met 80) that were repairable by MsrA. Repair of the oxidized methionines in cyt c restored the activity of cytochrome c oxidase and reduced cytochrome c peroxidase activity.. These results establish that MsrA deletion causes increased light scattering in mice exposed to HBO and they identify cyt c as oxidized in the knockout lenses. They also establish that MsrA can restore the in vitro activity of cyt c through its repair of PMSO. These results support the hypothesis that MsrA is important for the maintenance of lens transparency and provide evidence that repair of mitochondrial cyt c by MsrA could play an important role in defense of the lens against cataract formation.

Topics: Animals; Cataract; Cell Line; Cytochromes c; Disease Models, Animal; Gene Deletion; Humans; Hyperbaric Oxygenation; Lens, Crystalline; Light; Methionine; Methionine Sulfoxide Reductases; Mice; Mice, Knockout; Mitochondrial Proteins; Oxidation-Reduction; Oxidative Stress; Oxidoreductases; Scattering, Radiation; Spectrometry, Mass, Electrospray Ionization