Cytochrome c (cyt c), in addition to its function as an electron shuttle in respiratory chain, is able to perform as a pseudo-peroxidase with a critical role during apoptosis. Incubation of cyt c with an excess of hydrogen peroxide leads to a suicide inactivation of the protein, which is accompanied by heme destruction and covalent modification of numerous amino acid residues. Although steady-state reactions of cyt c with an excess of hydrogen peroxide represent non-physiological conditions, they might be used for analysis of the first-modified amino acid in in vivo. Here, we observed oxidation of tyrosine residues 67 and 74 and heme as the first modifications found upon incubation with hydrogen peroxide. The positions of the oxidized tyrosines suggest a possible migration pathway of hydrogen peroxide-induced radicals from the site of heme localization to the protein surface. Analysis of a size of folded fraction of cyt c upon limited incubation with hydrogen peroxide indicates that the early oxidation of amino acids triggers an accelerated destruction of cyt c. Position of channels from molecular dynamics simulation structures of cyt c points to a location of amino acid residues exposed to reactive oxidants that are thus more prone to covalent modification.

• MeSH
• cirkulární dichroismus MeSH
• cytochromy c chemie   genetika   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• koně MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• oxidace-redukce MeSH
• peroxid vodíku farmakologie   MeSH
• proteolýza MeSH
• simulace molekulární dynamiky MeSH
• stabilita proteinů MeSH
• tyrosin chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

By means of fluorescence microscopy the intracellular distribution of fluorescent drugs with different hydrophobicity (quinizarin, emodin and hypericin) was studied. Selective photoactivation of these drugs in precisely defined position (nuclear envelope) allowed moderately hydrophobic emodin enter the nucleus. Highly hydrophobic hypericin was predominantly kept in the membranes with no fluorescence observed in the nucleus. The redistribution of quinizarin, emodin and hypericin between lipids, proteins and DNA was studied in solutions and cells. Based on these results was proposed theoretical model of hydrophobic drugs' nuclear internalization after photo-activation. Molecular docking models showed that hypericin has the strongest affinity to P-glycoprotein involved in the cell detoxification. Presence of 10 μM quinizarin, emodin or hypericin increased P-glycoprotein function in U87 MG cells. Moreover, emodin pretreatment allowed quinizarin nuclear internalization without photo-activation, which was not the case for hypericin. The synergy of such pretreatment and photo-activation should lessen the drug doses with simultaneous increase of drug efficacy triggering cell apoptosis/necrosis.

• MeSH
• anthrachinony chemie   farmakologie   účinky záření   MeSH
• buněčné jádro metabolismus   účinky záření   MeSH
• DNA chemie   MeSH
• emodin chemie   farmakologie   účinky záření   MeSH
• gliom metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• LDL-cholesterol chemie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• P-glykoprotein metabolismus   MeSH
• perylen analogy a deriváty   chemie   farmakologie   účinky záření   MeSH
• sérový albumin chemie   MeSH
• simulace molekulového dockingu MeSH
• světlo MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH