Doctoral researcher: Daniel Trnka
Principle investigator: Christopher Horst Lillig
Co-supervisors: C. A. Helm, H. Antelmann
How does mitochondrial Grx2 protect from doxorubicin toxicity, cardiolipin peroxidation, and apoptosis?
Various cytostatic drugs induce apoptosis through the induction of mitochondrial ROS production. The anthracycline antibiotic doxorubicin/adriamycin, for instance, is able to redox cycle with complex I of the inner mitochondrial membrane that generates a semi-quinone radical that initiates a vicious cycle resulting in the production of superoxide anion radicals . Enzymatic degradation and Fenton chemistry convert superoxide to hydrogen peroxide and hydroxyl radicals. These ROS react directly with biomolecules and induce their cleavage, sulfoxidation, hydroxylation, peroxidation, or carbonylation. Cardiolipin is an essential phospholipid that participates in anchoring cytochrome C to the inner mitochondrial membrane. Oxidation and loss of cardiolipin leads to the release of cytochrome C and induction of apoptosis through caspase activation . Cardiomyocytes are especially vulnerable to these events and therefore limit the use of the drug. The oxidoreductase glutaredoxin 2 (Grx2) attenuates doxorubicin-induced apoptosis by preventing the oxidation and loss of cardiolipin . Cells lacking Grx2 are thus highly sensitized to doxorubicin treatment . Grx2 can complex an iron-sulfur cluster [5;6] and may participate in the regulation of mitochondrial iron homeostasis . This project aims at the identification and characterization of the mechanism that underlies Grx2's protective functions. First, potential protein interaction partners for Grx2 will be identified by proteomic techniques, for instance by trapping of substrates with mutant Grx2 unable to resolve the intermediate mixed disulfides that form during catalysis. Special emphasis will be put on membrane proteins such as subunits of complex I, that have been suggested to contain susceptible thiol groups important for reaction with quinon groups before . Second, the influence of Grx2 on the levels of redox-active free iron that is available for Fenton chemistry will be addressed in cells with silenced and elevated levels of Grx2 and mutants unable to bind the iron cofactor6. Third, we will analyze the effects of Grx2 on the extent and nature of cardiolipin oxidation. Fourth, we will investigate how cardiolipin oxidation changes the physico-chemical properties of the membrane and the attachment of cytochrome C to it. This PhD project would not be possible without the transdisciplinary approach this unique consortium offers. Expertise in cell biology, and protein (redox-)biochemistry is available in the group of project A1. Redox proteomics in project C1 (Antelmann), the analysis of cardiolipin species will be provided by associate member Schild (Magdeburg). Analytical methods are offered in projects B4 (Lalk/Kahlert) and C3 (Stöhr). Protein crystallography to study protein-substrate interactions will be provided by associate member Hinrichs (Greifswald). The analysis of the physico-chemical properties of the membrane and protein-membrane interactions requires the combined expertises of projects B1 (Helm), B2 (Scholz), and B3 (Brezesinski).
1 Davies, K.J., Doroshow, J.H.: J. Biol. Chem. (1986) 261, 3060.
2 Orrenius, S., Zhivotovsky, B. Cardiolipin oxidation sets cytochrome c free. Nat. Chem. Biol. (2005) 1, 188.
3 Enoksson, M., Potamitou Fernandes, A., Prast, S., Lillig, C.H., Holmgren, A., Orrenius, S.: Biochem. Biophys. Res. Commun. (2005) 327, 774.
4 Lillig, C.H., Lönn, M.E., Enoksson, M., Potamitou Fernandes, A., Holmgren, A.: Proc. Natl. Acad. Sci. U.S.A. (2004) 101, 13227.
5 Lillig, C.H., Berndt, C., Vergnolle, O., Lönn, M.E., Hudemann, C., Bill, E., Holmgren, A.: Proc. Natl. Acad. Sci. U.S.A. (2005) 102, 8168.
6 Berndt, C., Hudemann, C., Hanschmann, E.M., Axelsson, R., Holmgren, A., Lillig, C.H.: Antioxid. Redox Signal. (2007) 9, 151.
7 Lee, D.W., Kaur, D., Chinta, S.J., Rajagopalan, S., Andersen, J.K.: Antioxid. Redox Signal. (2009) 11, 2083.
8 Galkin, A., Meyer, B., Wittig, I., Karas, M., Schägger, H., Vinogradov, A., Brandt, U.: J. Biol. Chem. (2008) 283, 20907.
PD Dr. Christopher H. Lillig (Vice Speaker)
University Medicine Greifswald