Redox regulation
The group has extensively investigated the impact of redox biology and redox mechanisms within the context of cell signaling in yeast and mammalian cells for more than two decades.
Redox homeostasis, that is, the correct balance between the levels of ROS/RNS and antioxidant systems is essential for the proper functioning of cells. A wide variety of stimuli involving signal molecules (growth factors, cytokines and G-protein-coupled receptors) have been described that generate H2O2.
The focus of our research is a group of proteins known as "redoxins" (glutaredoxin, thioredoxin, peroxiredoxin) responsible for controlling the redox status of critical cysteine residues in target proteins and how they affect metabolism, signaling pathways, cell cycle and cell proliferation.
Our aim is to gain basic knowledge using experimental models, mainly tumoral cell lines in culture, and we expect our findings to shed light on the molecular and cellular basis of pathologies and to help optimize current preventive and therapeutic strategies.
Peroxiredoxin 6 (Prdx6)
Thioredoxin 1 (Trx1)
Glutaredoxin 1 (Grx1)
Projects and achievements
Redox-induced metabolic re-programming of hepatocarcinoma cells.
We
have shown that Trx and/or Grx are involved in redox modifications of targeted
cysteines, part of a widespread adaptive mechanism aimed at redistributing metabolic
fluxes between Glycolysis and its off-shooting pathways.
Trx1 and Grx1, are a hindrance for the antiproliferative action of NO, supporting the contention that weakening of thiolic antioxidant defenses stimulates the antiproliferative effects of NO in tumoral cells.
Trx, Nitric Oxide (NO), Sorafenib and cancer
The expression levels of Trx1 and TrxR1 followed the same trend as canonical EMT markers and its downregulation transformed advanced HCC cells into Sorafenib-sensitive cells. These findings support the idea that a combination of Sorafenib with thioredoxin inhibitors should be taken into account in the design of therapies against advanced HCC.
Catalytic mechanism of 1-Cys peroxiredoxins
GSH is a cofactor in the catalytic mechanism of mitochondrial yeast Prdx1 at concentration >100 times lower than normal without its redox state being modified during the process, and protects Prx1 from overoxidation. Moreover, Trx3 has deglutathionylating activity.
Important roles of Prdx6 in hepatocarcinoma cells
The cellular response to lack of PRDX6 is
graphically summarized in the figure. Therefore, our results support a role of PRDX6 in cell cycle control, proliferation, metabolism, mitochondrial function and autophagy.
We expect to describe whether the PLA2 activity through lipokines or the peroxidase activity through redox changes are responsible for these functions and whether the Grx/GSH system also plays a role. A functional connection between Prdx6 and the NOX system and a role in migration and invasiveness are also a matter of current concern.
Methodologies and Experimental Strategies
Proteomics
We have developed and optimized a Proteomics workflow to quantitatively analyze the differential global and Redox proteome and have applied it to the study of the role of redoxins in hepatocarcinoma cells.
The method has allowed for the discovery of target redox active cysteine residues in key proteins that affect metabolic fluxes, cell cycle, cell signaling, etc.
In vivo metabolic flux analysis ("Seahorse")
The use of the "Seahorse" methodology to measure in situ oxygen consumption rate and pH changes in culture medium of live cells has allowed us for the determination of glycolytic and respiratory metabolic fluxes in hepatocarcinoma cells with different redoxin loads and to ascertain the cause of mitochondrial dysfunction.
Flow cytometry
Flow cytomerric analyses have demonstrated cell cycle arrest at specific phases caused by changes in Prdx6 cellular levels.
Electron microscopy
Electron micrographs have evidenced the effect of lack of Prdx6 in Autophagy and mitochondrial dynamics.