UVM Theses and Dissertations
Format:
Print
Author:
Aesif, Scott William
Dept./Program:
Cell and Molecular Biology Program
Year:
2009
Degree:
Ph. D.
Abstract:
Protein S-glutathionylation is the conjugation of the low molecular weight antioxidant molecule glutathione to oxidized and reactive cysteine residues within proteins. Recently this post-translational event has been likened to O-phosphorylation given its specificity to oxidized and reactive cysteines, and its regulated reversibility. The resolution of S-glutathionylated cysteines is accomplished through members of the oxidoreductase family of enzymes, namely thioltransferases or glutaredoxins. Glutaredoxin-1 (Grx1) has been shown to be critical in regulating the deglutathionylation of a significant number of proteins critical to cellular signaling and structure. One signaling pathway determined to be regulated through S-glutathionylation of various pro-signaling subunits is the nuclear factor kappa-B (NF-[kappa]B) pathway. Known as a "master regulator" of immunity, the NF-[kappa]B pathway has been shown to regulate over one hundred genes involved in survival, inflammation, innate immunity, and adaptive immunity. Within the lung, the NF-[kappa]B pathway has been shown to be critical in responding to aspirated bacterial toxin lipopolysaccharide (LPS), the pollutant nitrogen dioxide (NO₂), and in the generation of allergic responses to inhaled antigen.
Given the relationship between protein S-glutathionylation and the NF-[kappa]B pathway, we sought to determine the consequences of genetically ablating or over expressing Grx1 with regard to pathogen-induced inflammatory responses in the lung. The studies presented in this thesis were aimed at frst defining a technique for determining patterns of protein S-glutathionylation in situ. Establishing this technique resulted in the observation that within the lung, S-glutathionylated proteins are present in the conducting airways, the pulmonary parenchyma, as well as alveolar macrophages, with the latter being the most significant source of S-glutathionylated proteins.
We next determined what impact genetic ablation and over expression of Grx1 would have on NF-[kappa]B induced pulmonary inflammation following LPS aspiration or infection with Psetrdomonas aeruginosa. Despite modestly impairing LPS induced inflammation, Grx1 ablation resulted in a significant impairment of alveolar macrophage maturation, activation, and phagocytosis following LPS stimulation. On infection with Pseudomonas aeruginosa however, ablation of Grx1 resulted in a marked survival advantage with significantly lower bacterial colony counts and cytokine production compared to wild type mice. Conversely, over expression of Grx1 within the airway epithelium resulted in significantly higher bacterial counts following infection.
Finally, in addressing the poorly understood mechanisms by which Grx1 expression is regulated at the level of transcription, we investigated the possibility that the NF-[kappa]B pathway could regulate Grxl expression in a potential feed forward mechanism leading to the propagation of inflammatory responses. Indeed, activation of the NF-[kappa]B pathway in alveolar epithelial cells and macrophages resulted in significant Grx1 protein induction. Conversely, inhibition of the NF-[kappa]B pathway significantly inhibited Grx1 protein induction following LPS stimulation of macrophages. Taken together these results suggest a Grxl and NF-[kappa]B axis for a feed forward mechanism of inflammation and potential new avenue for targeted therapeutic intervention.
Given the relationship between protein S-glutathionylation and the NF-[kappa]B pathway, we sought to determine the consequences of genetically ablating or over expressing Grx1 with regard to pathogen-induced inflammatory responses in the lung. The studies presented in this thesis were aimed at frst defining a technique for determining patterns of protein S-glutathionylation in situ. Establishing this technique resulted in the observation that within the lung, S-glutathionylated proteins are present in the conducting airways, the pulmonary parenchyma, as well as alveolar macrophages, with the latter being the most significant source of S-glutathionylated proteins.
We next determined what impact genetic ablation and over expression of Grx1 would have on NF-[kappa]B induced pulmonary inflammation following LPS aspiration or infection with Psetrdomonas aeruginosa. Despite modestly impairing LPS induced inflammation, Grx1 ablation resulted in a significant impairment of alveolar macrophage maturation, activation, and phagocytosis following LPS stimulation. On infection with Pseudomonas aeruginosa however, ablation of Grx1 resulted in a marked survival advantage with significantly lower bacterial colony counts and cytokine production compared to wild type mice. Conversely, over expression of Grx1 within the airway epithelium resulted in significantly higher bacterial counts following infection.
Finally, in addressing the poorly understood mechanisms by which Grx1 expression is regulated at the level of transcription, we investigated the possibility that the NF-[kappa]B pathway could regulate Grxl expression in a potential feed forward mechanism leading to the propagation of inflammatory responses. Indeed, activation of the NF-[kappa]B pathway in alveolar epithelial cells and macrophages resulted in significant Grx1 protein induction. Conversely, inhibition of the NF-[kappa]B pathway significantly inhibited Grx1 protein induction following LPS stimulation of macrophages. Taken together these results suggest a Grxl and NF-[kappa]B axis for a feed forward mechanism of inflammation and potential new avenue for targeted therapeutic intervention.