cytochrome-c-t and Cataract

α-Crystallin is a member of the small heat-shock protein (sHSP) family and consists of two subunits, αA and αB. Both αA- and αB-crystallin act as chaperones and anti-apoptotic proteins. Previous studies have identified the peptide (70)KFVIFLDVKHFSPEDLTVK(88) in αA-crystallin and the peptide (73)DRFSVNLDVKHFSPEELKVK(92) in αB-crystallin as mini-chaperones. In the human lens, lysine 70 (Lys(70)) of αA and Lys(92) of αB (in the mini-chaperone sequences) are acetylated. In this study, we investigated the cellular effects of the unmodified and acetyl mini-chaperones. The αA- and αB-crystallin peptides inhibited stress-induced aggregation of four client proteins, and the αA-acetyl peptide was more effective than the native peptide against three of the client proteins. Both the acetyl and native crystallin peptides inhibited stress-induced apoptosis in two mammalian cell types, and this property was directly related to the inhibition of cytochrome c release from mitochondria and the activity of caspase-3 and -9. In organ-cultured rat lenses, the peptides inhibited calcimycin-induced epithelial cell apoptosis. Intraperitoneal injection of the peptides inhibited cataract development in selenite-treated rats, which was accompanied by inhibition of oxidative stress, protein insolubilization, and caspase activity in the lens. These inhibitory effects were more pronounced for acetyl peptides than native peptides. A scrambled αA-crystallin peptide produced no such effects. The results suggest that the α-crystallin chaperone peptides could be used as therapeutic agents to treat cataracts and diseases in which protein aggregation and apoptosis are contributing factors.

Topics: Adult; alpha-Crystallin A Chain; alpha-Crystallin B Chain; Animals; Apoptosis; Caspase 3; Caspase 9; Cataract; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Cytochromes c; Disease Models, Animal; Epithelial Cells; Humans; Inhibitor of Apoptosis Proteins; Molecular Chaperones; Rats; Rats, Sprague-Dawley

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

In this study, the preparation, stability and anti-cataract effect of cationic freeze-dried liposomes containing cytochrome c, along with nicotinamide and adenosine, are described.. Cytochrome c-loaded cationic liposomes (CC-L) were prepared by the thin-layer evaporation technique and lyophilized to obtain freeze-dried cytochrome c liposomes (CC-F). The influence of the preparation components on the liposomal encapsulation efficiency and the stability were studied. The anti-cataract effect of the CC-F was demonstrated through attenuating lens opacity development with slit lamp examination in rats with selenite-induced cataract.. Our study indicates that: (1) the liposomal encapsulation efficiency increased with increasing phosphatidylcholine content and reduced in the presence of stearylamine. Moreover, optimal encapsulation efficiency was obtained at an appropriate ratio of phosphatidylcholine to cholesterol; (2) CC-F was stable for at least 12 months at 4 degrees C; (3) satisfactory improvements in lens opacity were shown in the cytochrome c-treated groups, especially for the CC-F-treated group with the decreased percentage of lens opacity at about 28% at the final examination.. CC-F were shown to be stable superior ophthalmic carriers and were able to markedly retard the onset of cataract development.

Topics: Animals; Cataract; Cytochromes c; Drug Carriers; Drug Stability; Freeze Drying; Liposomes; Ophthalmic Solutions; Rats; Rats, Sprague-Dawley; Sodium Selenite

An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 mumol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.

Topics: Animals; Base Sequence; Cataract; Cell Line; Cytochromes c; DNA; DNA Damage; Epithelial Cells; Gene Expression; Gene Expression Profiling; Lens, Crystalline; Mice; Mitochondria; Oligonucleotide Array Sequence Analysis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium Selenite