UVM Theses and Dissertations
Format:
Print
Author:
Randall, Matthew J.
Dept./Program:
Pathology
Year:
2012
Degree:
MS
Abstract:
Exposure to cigarette smoke (CS) is associated with lung cancer, asthma, chronic obstructive pulmonary disease and emphysema. Acrolein, a prominent constituent of CS, is known to illicit many of the same physiological effects as whole CS. As an electrophile, the alpha, beta-unsaturated aldehyde, acrolein, forms adducts with cellular nucleophiles such as, histidine, lysine, and cysteine amino acid residues. It has been suggested that the active site selenocysteine of thioredoxin reductase 1 (TrxR1) is highly sensitive to adduction of electrophiles which can cause a gain-of-function and promote cellular apoptosis. This modified TrxR1 is known as a selenium-compromised thioredoxin reductase-derived apoptotic protein (SecTRAP). The SecTRAP may therefore have profound effects on cellular functions observed in smoking related diseases.
Using purified semisynthetic mitochondrial TrxR (mTrxR-GCUG) or a truncated variant lacking the last three amino acid residues (mTrxRdelta3), we first assessed the effects of acrolein adduction on the enzymatic activity of TrxR. We demonstrated that adduction of acrolein to mTrxR-GCUG decreases its reductase activity and increases its consumption of NADPH. Further, we addressed whether the alterations in TrxR activity have functional consequences for the lung epithelium using immortalized human bronchiolar epithelial (HBE1) cells. To identify the acrolein-mediated effects specific to TrxR1, HBEl cells were supplemented with sodium selenite to increase TrxR1 levels or transfected with siRNA against TXNRD1 to knock down TrxR1. Analyses were performed to assess protein adduction of acrolein, TrxR activity, Thioredoxin 1 oxidation, MAPK phosphorylation, antioxidant gene expression, cytokine release, and cell viability.
We discovered a novel and significant role for TrxR1 in acrolein-mediated MAPK phosphorylation. The physiological effects regarding gene expression, cytokine release, and cell viability were not dependent of TrxR1-mediated MAPK phosphorylation. We then assessed the reversibility of protein-acrolein adducts. Using biotin hydrazide labeling and neutravidin chromatography, we assessed the reversibility of these adducts in HBE1 cells at varying times after acrolein treatment. Analyses were made addressing TrxR activity, G5H levels, proteasomal degradation, and loss of protein-acrolein adduct. We illustrated that protein-acrolein adducts are not being degraded by the 26S proteasome and that the time-dependent loss of these adducts may depend on cellular GSH status.
Collectively, these studies illustrate that acrolein-induced phosphorylation of MAPKs, and specifically JNK1, is mediated by TrxR1 independently of Trx1 oxidation state. The physiological effects of this phenomenon are yet to be determined. We also suggest that reversible cysteine alkylation may be an unrecognized signaling mechanism similar to S-glutathionylation and S-nitrosylation. Further exploration of these studies can initiate the development of therapeutics for cigarette smoke related diseases.
Using purified semisynthetic mitochondrial TrxR (mTrxR-GCUG) or a truncated variant lacking the last three amino acid residues (mTrxRdelta3), we first assessed the effects of acrolein adduction on the enzymatic activity of TrxR. We demonstrated that adduction of acrolein to mTrxR-GCUG decreases its reductase activity and increases its consumption of NADPH. Further, we addressed whether the alterations in TrxR activity have functional consequences for the lung epithelium using immortalized human bronchiolar epithelial (HBE1) cells. To identify the acrolein-mediated effects specific to TrxR1, HBEl cells were supplemented with sodium selenite to increase TrxR1 levels or transfected with siRNA against TXNRD1 to knock down TrxR1. Analyses were performed to assess protein adduction of acrolein, TrxR activity, Thioredoxin 1 oxidation, MAPK phosphorylation, antioxidant gene expression, cytokine release, and cell viability.
We discovered a novel and significant role for TrxR1 in acrolein-mediated MAPK phosphorylation. The physiological effects regarding gene expression, cytokine release, and cell viability were not dependent of TrxR1-mediated MAPK phosphorylation. We then assessed the reversibility of protein-acrolein adducts. Using biotin hydrazide labeling and neutravidin chromatography, we assessed the reversibility of these adducts in HBE1 cells at varying times after acrolein treatment. Analyses were made addressing TrxR activity, G5H levels, proteasomal degradation, and loss of protein-acrolein adduct. We illustrated that protein-acrolein adducts are not being degraded by the 26S proteasome and that the time-dependent loss of these adducts may depend on cellular GSH status.
Collectively, these studies illustrate that acrolein-induced phosphorylation of MAPKs, and specifically JNK1, is mediated by TrxR1 independently of Trx1 oxidation state. The physiological effects of this phenomenon are yet to be determined. We also suggest that reversible cysteine alkylation may be an unrecognized signaling mechanism similar to S-glutathionylation and S-nitrosylation. Further exploration of these studies can initiate the development of therapeutics for cigarette smoke related diseases.