Cellular Redox Imbalance and Changes of Protein S-glutathionylation Patterns Are Associated with Senescence Induced by Oncogenic H-Ras

Abstract

H-Ras oncogene requires deregulation of additional oncogenes or inactivation of tumor suppressor proteins to increase cell proliferation rate and transform cells. In fact, the expression of the constitutively activated H-RasV12 induces cell growth arrest and premature senescence, which act like barriers in pre-neoplastic lesions. In our experimental model, human fibroblasts transfected with H-RasV12 show a dramatic modification of morphology. H-RasV12 expressing cells also show premature senescence followed by cell death, induced by autophagy and apoptosis. In this context, we provide evidence that in H-RasV12 expressing cells, the premature senescence is associated with cellular redox imbalance as well as with altered post-translation protein modification. In particular, redox imbalance is due to a strong reduction of total antioxidant capacity, and significant decrease of glutathione level. As the reversible addition of glutathione to cysteinyl residues of proteins is an important post-translational regulative modification, we investigated S-glutathionylation in cells expressing active H-Ras. In this contest we observed different S-glutathionylation patterns in control and H-RasV12 expressing cells. Particularly, the GAPDH enzyme showed S-glutathionylation increase and significant enzyme activity depletion in H-Ras V12 cells. In conclusion, we proposed that antioxidant defense reduction, glutathione depletion and subsequent modification of S-glutathionylation of target proteins contribute to arrest cell growth, leading to death of fibroblasts expressing constitutively active H-Ras oncogene, thus acting as oncogenic barriers that obstacle the progression of cell transformation.

Glutathione compartmentalization and its role in glutathionylation and other regulatory processes of cellular pathways

“…However, it also operates as a biological redox switch in the regulation of signal transduction and metabolic pathways [20,72]. Indeed, S-glutathionylation occurs on protein kinases, checkpoints of cell cycle regulation and death pathways, as well as on proteins involved in glycolysis/energy metabolism regulation, antioxidant enzymes, and on cytoskeletal and chaperone proteins [20,67,[73][74][75][76][77][78].…”

Effect of High Glucose-Induced Oxidative Stress on Paraoxonase 2 Expression and Activity in Caco-2 Cells

“…Samples were analyzed using Guava easyCyteflow cytometer (Merck Millipore, Darmstadt, Germany). For each sample, 5000 events were acquired [44]. Annexin V-FITC is detected as a green fluorescence and propidium iodide is detected as a red fluorescence.…”

A possible S‐glutathionylation of specific proteins by glyoxalase II: An in vitro and in silico study

“…Previously, S‐glutathionylation was considered as a nonspecific response to cellular oxidative status to protect cysteines from irreversible oxidation. Recently, it has been proposed as a regulatory and selective posttranslational protein modification in response to specific physiological redox signaling …”

“…Recently, it has been proposed as a regulatory and selective posttranslational protein modification in response to specific physiological redox signaling. [16][17][18] Glo2, similarly to GSTPs, produces a thiolate anion at the active site (GS − ) 7 so we decided to investigate a possible S-glutathionylation reaction between Glo2 and actin, a well-known protein that undergoes this posttranslational modification, and between Glo2 and malate dehydrogenase (MDH) as, in previous experiments by our group, a co-purification with MDH was observed during the isolation of mitochondrial Glo2 from rat liver (Table S1).…”