Abstract
Heart failure is the most common cause of morbidity and hospitalization in the western civilization. Protein phosphatases play a key role in the basal cardiac contractility and in the responses to β-adrenergic stimulation with type-1 phosphatase (PP-1) being major contributor. We propose here that formation of transient disulfide bridges in PP-1α might play a leading role in oxidative stress response. First, we established an optimized workflow, the so-called “cross-over-read” search method, for the identification of disulfide-linked species using permutated databases. By applying this method, we demonstrate the formation of unexpected transient disulfides in PP-1α to shelter against over-oxidation. This protection mechanism strongly depends on the fast response in the presence of reduced glutathione. Our work points out that the dimerization of PP-1α involving Cys39 and Cys127 is presumably important for the protection of PP-1α active surface in the absence of a substrate. We finally give insight into the electron transport from the PP-1α catalytic core to the surface. Our data suggest that the formation of transient disulfides might be a general mechanism of proteins to escape from irreversible cysteine oxidation and to prevent their complete inactivation.
Highlights
The formation of transient disulfides within KIM-like tyrosine phosphatases is linked with the reversible inactivation of the phosphatase activity[46]
Our data demonstrate that PP-1α is more robust to withstand higher levels of redox stress − 50% inactivation is observed at 500 μM H2O2 for PP-1α, but already at 150 μM H2O2 for the KIM-like tyrosine phosphatases
Previous study showed that oxidative stress led to formation of stable complexes containing PP-1α, GADD34, elF2a and TDP-43 and enhancing the substrate binding[47]
Summary
Live-cell imaging of NRCMs. NRCMs were isolated at postnatal day 1–3 and cultured in a 6-well plate for 3–4 days. 90 min before live-cell imaging, NRCMs were incubated with 0.1, 1 or 10 mM H2O2 in the climate chamber of the Olympus fluorescence microscope (37 °C, 5% CO2). The video frames were recorded with 57.37% lamp intensity every 20 min with an exposure time of 20 ms for 24 h. A matlab based script was used to generate heat maps of the mean contraction for each given H2O2 concentrations[18]. A resolution of 0.6442 μm/pixel was used and dead floating cells were filtered. The changes in the morphology of NRCMs were analysed with Image
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