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Proapoptotic activity of cytochrome c in living cells: effect of K72 substitutions and species differences

  • Rita V. Chertkova
  • George V. Sharonov
  • Alexei V. Feofanov
  • Ol’ga V. Bocharova
  • Ramil F. Latypov
  • Boris V. Chernyak
  • Alexander S. Arseniev
  • Dmitry A. Dolgikh
  • Mikhail P. Kirpichnikov
Article

Abstract

Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) ≫ h(K72W) > m(K72W). As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c ≈ horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.

Keywords

Apoptosis Cytochrome c Mutagenesis Electroporation Fluorescence microscopy Confocal spectral imaging 

Abbreviations

Apaf-1

Apoptotic protease-activating factor

AnV

Recombinant human annexin V conjugated with R-phycoerythrin

CSI

Confocal spectral imaging

FCS

Fetal calf serum

h(K72A), h(K72E), h(K72L), and h(К72W)

Horse heart cytochrome c mutants K72A, K72E, K72L, and К72W, respectively

m(K72W)

Murine cytochrome c mutant К72W

PI

Propidium iodide

Rh

Rhodamine 123

TR

Tetramethylrhodamine

Notes

Acknowledgments

We gratefully acknowledge Professor V. P. Skulachev for his encouragement and fruitful discussions. Thе research was supported by the RAS MCB Programme and by the grant SS-1061.2008.4.

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Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Rita V. Chertkova
    • 1
  • George V. Sharonov
    • 1
  • Alexei V. Feofanov
    • 1
    • 2
  • Ol’ga V. Bocharova
    • 1
  • Ramil F. Latypov
    • 3
    • 4
  • Boris V. Chernyak
    • 5
  • Alexander S. Arseniev
    • 1
  • Dmitry A. Dolgikh
    • 1
    • 2
  • Mikhail P. Kirpichnikov
    • 1
    • 2
  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Bioengineering Department, Biological FacultyMoscow State UniversityMoscowRussia
  3. 3.Basic Science DivisionFox Chase Cancer CenterPhiladelphiaUSA
  4. 4.Amgen IncSeattleUSA
  5. 5.A.N.Belozersky Institute of Physico-Chemical BiologyMoscow State UniversityMoscowRussia

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