Background: Extensive recent experimental work has established hydrogen peroxide (H2O2) as second messenger, able to mediate intracellular signals via selective oxidation of cysteine residues in signaling proteins (redox signaling). However, the exact molecular mechanisms underlying these pathways as well as the details of the reaction between H2O2 and cysteines remain poorly understood. Existing experimental evidence from our as well as from other laboratories indicates that redox-active iron may be an important mediator of H2O2-induced oxidation of cysteines but this area is poorly understood and needs further investigation.
Aim: To determine the role of intracellular labile iron in redox signal transductions.
Research strategy: In the initial phase of the work, we will establish cell lines with the ability of conditional expression of proteins that control intracellular labile ironlevels.
These cells will be used by all participating groups in order to investigate the role of iron in the process of H2O2- and TNFa-induced:
- apoptotic signals,
- oxidation of thioredoxins and glutaredoxins which control the activity of the MAP3K apoptosis signaling-regulated kinase 1 (ASK-1), and
- oxidation of specific cysteines in dual specificity phosphatases (DUSPs).
In addition, we will develop mass spectrometry based proteomic approaches for global identification of proteins containing oxidized cysteines and will determine the structural elements on protein targets implicated in the reactivity of specific cysteines.
Expected results: Successful completion of this study will contribute to better understanding of the molecular mechanism responsible for the interaction of H2O2 with specific cysteine residues and the involvement of iron in this interaction. We also anticipate the identification of signaling proteins in which the oxidation of cysteine residues is dependent on redox-active iron.