Insights into the HyPer biosensor as molecular tool for monitoring cellular antioxidant capacity

Abstract

Aerobic metabolism brings inexorably the production of reactive oxygen species (ROS), which are counterbalanced by intrinsic antioxidant defenses avoiding deleterious intracellular effects. Redox balance is the resultant of metabolic functioning under environmental inputs (i.e. diet, pollution) and the activity of intrinsic antioxidant machinery. Monitoring of intracellular hydrogen peroxide has been successfully achieved by redox biosensor advent; however, to track the intrinsic disulfide bond reduction capacity represents a fundamental piece to understand better how redox homeostasis is maintained in living cells.In the present work, we compared the informative value of steady-state measurements and the kinetics of HyPer, a H2O2-sensitive fluorescent biosensor, targeted at the cytosol, mitochondrion and endoplasmic reticulum. From this set of data, biosensor signal recovery from an oxidized state raised as a suitable parameter to discriminate reducing capacity of a close environment. Biosensor recovery was pH-independent, condition demonstrated by experiments on pH-clamped cells, and sensitive to pharmacological perturbations of enzymatic disulfide reduction. Also, ten human cell lines were characterized according their H2O2-pulse responses, including their capacity to reduce disulfide bonds evaluated in terms of their migratory capacity.Finally, cellular migration experiments were conducted to study whether migratory efficiency was associated with the disulfide reduction activity. The migration efficiency of each cell type correlates with the rate of signal recovery measured from the oxidized biosensor. In addition, HyPer-expressing cells treated with N-acetyl-cysteine had accelerated recovery rates and major migratory capacities, both reversible effects upon treatment removal. Our data demonstrate that the HyPer signal recovery offers a novel methodological tool to track the cellular impact of redox active biomolecules.

HyPer as a tool to determine the reductive activity in cellular compartments

“…The obtained results indicate that cancer cells exhibit significantly higher reduction rates. This is consistent with previously published data of HyPer-based research [ 20 ] and the fact that cancer cells tend to overexpress components of the Trx system, rendering the Trx system a promising target for anticancer interventions [ 34 , 58 ]. The protocol established in our study could readily be adapted to high-throughput cellular screens to search for irreversible inhibitors of the Trx system directly in cells, a task typically performed in vitro at present [ 59 ].…”

“…The disulfide bond formed then becomes susceptible to reduction by the reducing systems of the cell. Lamentably, astoundingly little attention has hitherto been given to the reductive half-reaction, with the exception of only a few independent studies [ 20 , 37 , 38 ]. We consider this a serious omission, since understanding the mechanisms that govern this reaction is crucial to correctly interpreting the biosensor signal, determining whether an increase in the biosensor signal is due to elevated H 2 O 2 levels or a shortage of reducing equivalents (e.g.…”

“…Hence, monitoring HyPer1 fluorescence in various compartments of living human cells is widely used for following intracellular H 2 O 2 levels in function of time [ [17] , [18] , [19] ]. In addition, since the redox-active thiol groups of HyPer1 are targets not only for H 2 O 2 , but also for cellular oxidoreductases, this biosensor has recently proven to be a tool for monitoring the disulfide reducing activity in cells [ 20 ]. In the current study, we aimed to elucidate which thiol-reducing system (Trx/TrxR- or Grx/GSH-dependent) is responsible for HyPer1 reduction in the cytosol, nucleus, and mitochondria of human cells and developed a protocol to quantify the activity of these systems in living cells after the induction of transient oxidative stress.…”

Biological Redox Impact of Tocopherol Isomers Is Mediated by Fast Cytosolic Calcium Increases in Living Caco-2 Cells

“…The HyPer biosensor was introduced into the cytoplasm of Caco-2 cells by infection with adenovirus at 1:100 dilution. Adeno-particle production has been explained in detail by our group [13]. Briefly, cyto-HyPer cDNA (Evrogen, Moscow, Russia) was subcloned into the commercial adenoviral vector pAdEasy-RFP using conventional molecular biology techniques, and homologous recombination was done using BJ5183 cell transformation.…”

Human umbilical artery endothelial cells from Large‐for‐Gestational‐Age newborn have increased antioxidant efficiency and gene expression

“…Following the observation of the cytopathic effects (CPE), usually after 14-21 days, the cells were harvested and subjected to three freeze-thaw cycles, followed by centrifugation to remove cellular debris, and the resulting supernatant (2 ml) used to infect a 10 cm dish of 90% confluent AdHek cells. After the observation of CPEs, normally after 2-3 days, viral particles were purified and expanded by infecting 10 plates of AdHek cells (Hernández et al, 2018).…”