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Digital archive of theses discussed at the University of Pisa


Thesis etd-02062008-125259

Thesis type
Tesi di dottorato di ricerca
Thesis title
Mechanisms of molecular signaling mediated by reversible thiol oxidation
Academic discipline
Course of study
Relatore Prof.ssa Del Corso, Antonella
  • glutathione
  • GSH
  • lens epithelial cells
  • redox homeostasis
  • redox signaling potential
  • redox state
  • ROS
  • signaling molecules
Graduation session start date
Release date
Every cell is characterized by a reduction-oxidation (redox) state or redox homeostasis that is currently defined by the ratio between GSH and GSSG. This state is determined by the balance between the concentration of Reactive Oxygen Species (ROS) and the antioxidant intracellular buffering capacity (deriving from the combination of low molecular scavengers and enzymatic systems). If ROS production exceeds the cellular buffering capacity, direct damage of proteins, lipids and DNA will ensue, leading to a compromise of cellular and organ physiology and is implicated in pathologies, aging and apoptosis. ROS at moderate concentrations, however, can act as a physiological signal by modulating normal cellular events including DNA synthesis, enzymatic activity, selective gene expression and regulation of the cell cycle. In this acceptation the redox state is increasingly recognized as a primary regulator of cellular growth and development.
The tripeptide glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) is quantitatively the most important intracellular thiol redox buffer and also serves as a major reservoir for the redox labile sulfur amino acid cysteine. The transsulfuration pathway constitutes the major route of cysteine biosynthesis.
The transsulfuration and glutathione biosynthesis pathways are logical targets for redox control since the end products of these pathways are involved in redox homeostasis.
Results obtained until now in this research did not allow to detect in cultured lens epithelial cells the GSH extrasynthesis observed in intact lenses when the gamma-glutamyl transferase activity was inhibited by addition of serine borate. We investigated the possible lack of precursors and/or energy sources in epithelial cells with respect to intact lens, in which the epithelial monolayer is anyway supported by the bulk of fibers acting at least as reservoir of precursors. Therefore glutathione levels were measured in different experiments in which epithelial cells were incubated in the presence of glutathione precursors (L-Cys, L-Gln, L-Gly) and glucose as energy source.
In these conditions, when epithelial cells were treated with serine borate, an increase of GSH was indeed observed with respect to control cells incubated in the absence of serine borate. The significance of the observed GSH increase (approximately 20% of the initial value measured on 6 samples) is at present under investigation.
We have previously reported that, in intact cultured lens, the GSH extra synthesis induced by serine borate can be abolished by Propargylglycine. The rationale of such an effect is the inhibition exerted this compound on Cystathionine γ-lyase (CGL), a key enzyme of the transsulfuration pathway considered the limiting factor in free Cys production. The relevance of transsulfuration pathway in Cys supply for GSH extra synthesis is also supported by the significant increase in the level of CGL activity measured in crude extracts obtained from lenses cultured in the presence of serine borate.
Lens epithelial cells respond with GSH extra synthesis to GGT inhibition, as observed for intact lens. The external requirement of energy supply necessary in epithelial cells is conceivably circumvented in the intact lens by the presence of the bulk fibers.
Further evidence is offered for the involvement of transsulfuration pathway on the Cys replenishment necessary for GSH synthesis, through Cystathionine γ-lyase activity.
The results of the present work support the use of epithelial cells as a suitable model for the study of the GSH turnover in the lens.
The apparent regulatory role of CysGly in glutathione homeostasis needs to be more precisely defined also in light of possible connections between the gamma-glutamyl cycle and the transsulfuration pathway.