Year: 2002
Type: dissertation
Abstract: Reactive oxygen species (ROS) are now recognized as important mediators of the cellular responses to environmental changes. Reversible protein phosphorylation appears to be one of the major intracellular signal transduction mechanisms modulated by ROS. Calcineurin is a protein serine/threonine phosphatase involved in calcium-dependent signaling. In this work the mechanisms whereby calcineurin activity is modulated by ROS were investigated. H2O2 in submillimolar concentrations inhibited calcineurin. This inhibition was partly reversible by DTT and thioredoxin and involved oxidation of protein cysteines. In tissue and cell homogenates superoxide, but not other ROS was found to be an effective calcineurin inhibitor. Inhibition of calcineurin activity by superoxide was calcium-dependent. NO antagonized superoxide action on calcineurin. By modification of calcineurin isolation procedure aiming to avoid enzyme oxidation milligram amounts of highly active porcine calcineurin were isolated. Porcine calcineurin was very sensitive to inhibition by superoxide in a calcium and calmodulin-dependent manner. NO protected calcineurin against inhibition by superoxide but not by H2O2. Superoxide-inhibited calcineurin was reactivated by ascorbate and Fe2+/ascorbate suggesting oxidation of Fe2+ in the enzyme active center as the mechanism of superoxide inhibition of calcineurin. Metal analysis and EPR spectroscopy confirmed the hypothesis that native, active calcineurin has Fe2+-Zn2+ binuclear center in the enzyme active site and oxidative inactivation of the enzyme results in its conversion to Fe3+-Zn2+ center and partial Fe release from the enzyme. Treatment of Jurkat T-lymphoma cells with oxidants resulted in inhibition of calcineurin activity and blockade of calcineurin-NFAT signaling cascade. In addition, endogenous ROS generation in activated macrophages caused calcineurin inhibition, providing evidence that calcineurin could be an endogenous effector of ROS-mediated signaling.
Source: KOPS (University of Konstanz)
Author: Dmitry Namgaladze
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W52842453