An oxidative shield surrounds the endoplasmic reticulum (ER) and topologically predicts a privileged region for signaling Articles uri icon

publication date

  • November 2023

start page

  • S10

volume

  • 208, Supplement 1

International Standard Serial Number (ISSN)

  • 0891-5849

abstract

  • Studies upon reactive oxygen species over the last decades have shown the importance of H2O2 as secondary messenger in cell signaling. The modulation of this molecule in time, concentration and location is considered to be a key determinant for specificity. Such would be the case at the interface between the ER and the cytosol, as the outflow of H2O2 through Aquaporin 11 (AQP11) should reach particular proteins while being prevented of modifying others. Consequently, the development of a detailed topography of H2O2 reactivity nearby this cell compartment is of outmost interest to decipher how the signal can be regulated.

    To map H2O2 distribution in this locale, we have expressed the genetically encoded H2O2-sensitive HyPer probe targeted either to the cytosol (Cyto) or to specific regions of the external leaflet of the ER of HeLa cells, including the AQP11 cytosolic gate (A11- H1). Using this strategy, we have detected a shift in the basal ratio of A11-H1 resulting in a more reduced profile to that of Cyto, opposing initial expectations. Dithiothreitol (DTT) addition failed to induce further downward variations of Cyto to reach the level of activation of A11-H1. Moreover, kinetics of the response of the probes upon exogenous H2O2 boluses and subsequent DTT reduction while varying cytosolic redox conditions suggested that HyPer can be terminally oxidized, resulting in a decrease of its ratio rather than an increase.

    Taken together, our data indicate that HyPer presents a characteristic fluorescent profile at the high oxidative ranges achieved in the vicinity of the cytosolic face of the ER. While these results confirm that regional production of H2O2 is a foremost element for specificity, they also imply that careful analyses of complete cellular scenario should be performed in order to distinguish between either a reducing or a profoundly oxidative area.

subjects

  • Biology and Biomedicine