The biological roles of glutathione transferase Omega 1

Abstract

Glutathionylation is the reversible redox modification of protein thiols by disulphide formation with glutathione. Glutathionylation can alter protein structure and activity in response to changes in the oxidation state of the protein, thus modulating protein stability. The forward reaction is largely spontaneous while the reverse reaction (deglutathionylation) is predominantly catalysed by the Glutaredoxin (Grx) family of thioltransferases. Glutathione transferase Omega 1 (GSTO1-1) is an atypical glutathione transferase that has minimal functional resemblance with other members of the superfamily. GSTO1-1 has previously been shown to have high thioltransferase activity like glutaredoxins. Interestingly, GSTO1-1 has been reported to be differentially expressed in neurodegenerative diseases. Although the studies reporting these differences speculate on the GST-like activity of GSTO1-1, it is evident from data published by our laboratory that the primary role of GSTO1-1 is yet to be identified. This study investigated the role of GSTO1-1 in the glutathionylation cycle. Here, we show that human GSTO1-1, with a unique conserved cysteine at its active site, can catalyse the deglutathionylation of protein thiols in vitro and in cell lines. The kinetics of the catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in cell lines by means of immunoblotting and immunoprecipitation. We generated stable GSTO1-1 transfectants in T47-D breast cancer cells which are devoid of endogenous GSTO1-1. The over-expression of GSTO1-1 in these cells resulted in a global abatement of protein glutathionylation. Furthermore, we demonstrated that a mutation in the active cysteine residue (Cys-32) ablates the deglutathionylating activity, confirming the role of GSTO1-1 as a redox switch in regulating protein post translational modification. Mass spectrometry revealed four deglutathionylated targets of GSTO1-1, of which β-actin was validated by extensive immunoprecipitation studies and the physiological impact of deglutathionylated β-actin was confirmed by immunostaining. This study introduces GSTO1-1 as a novel member of the family of deglutathionylating enzymes which is currently restricted to glutaredoxins and sulfiredoxins and identifies specific proteins targeted by GSTO1-1 in cells. Additionally, we have developed and employed a novel and rapid method to quantify global glutathionylation in vitro to confirm the catalytic role of GSTO1-1 in the glutathionylation cycle. GSTO1-1 has been investigated in relation to a number of biologically and clinically significant pathways and disorders including drug resistance, Alzheimer’s disease, Parkinson’s disease, the action of anti-inflammatory drugs and susceptibility to chronic obstructive pulmonary disease (COPD). Since glutathionylation has also been implicated in the pathology of Alzheimer’s disease, Parkinson’s disease, COPD and inflammation, it is proposed that GSTO1-1 dependent glutathionylation and/or deglutathionylation could be a common factor. The data gathered from the initial phase of the project directed us to determine whether GSTO1-1 is required for inflammatory signalling in phagocytic immune cells such as macrophages. Inflammatory stimulants such as bacterial lipopolysaccharide (LPS) have been shown to induce the generation of reactive oxygen species (ROS) through the activation of Toll like receptor 4 (TLR4) and the recruitment of downstream signalling proteins resulting in the subsequent induction of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α and ROS generating NADPH oxidase 1 (NOX1). Following from the previously reported involvement of glutathione GSTO1-1 in the secretion of IL-1β and our recent discovery of its deglutathionylation activity, we have identified a novel role for GSTO1-1 in regulating the generation of ROS following LPS activation of the TLR4 pro-inflammatory cascade. We discovered that J774.1A macrophages deficient in GSTO1-1 do not respond to LPS and fail to elicit pro-inflammatory responses including the generation of ROS via NADPH oxidase 1 and the expression of pro-IL-1β. The present data also show that the suppression of several antioxidant enzymes (catalase, glutathione peroxidase, glutamate-cysteine ligase) that normally protect against the effects of oxidative stress in LPS treated J774.1A cells is dependent on the presence of GSTO1-1. In order to confirm that the redox events unfolding in the presence of GSTO1-1 were due to its catalytic activity, we tested the GSTO1-1 inhibitor ML175 on wildtype J774.1A macrophages. The production of ROS and the suppression of antioxidant enzymes after LPS stimulus were blocked significantly by pre-treating cells with ML175, clearly mimicking the response of GSTO1-1 knockdown cells. Taken together, our data demonstrate the significant attenuation of ROS in GSTO1-1 deficient cells, thus identifying a novel component of the ROS production pathway in LPS activated macrophages and placing GSTO1-1 in the TLR4 signalling pathway, upstream of NF-κB. TLR4 ligands such as LPS modulate the metabolic activity of macrophages, skewing cells towards a more glycolytic phenotype which is characterized by an increase in metabolic flux through the pentose phosphate pathway (PPP) and lower oxygen consumption (OCR). We show that the glycolytic switch is significantly attenuated in GSTO1-1 deficient macrophages, which we propose results from an upstream block in the TLR4 signalling pathway. Our studies on GSTO1-1 deficient cells demonstrate that AMPKα, a key metabolic stress regulator is maintained in a phosphorylated (active) state in macrophages after LPS stimulation, supporting its anti-inflammatory role. In addition, Gsto1 knockdown cells were unable to induce HIF1α in response to LPS, thus indicating their failure to acquire a glycolytic phenotype. This was confirmed by their low extracellular acidification rate (ECAR). The findings in the cell line studies translated well in vivo as the Gsto1-/- mice failed to elicit an adequate inflammatory response when injected with sub-lethal and lethal doses of LPS intra-peritoneally. Subsequent studies focused on identifying the target(s) of GSTO1-1 in the TLR4 pathway. We have successfully placed GSTO1-1 upstream of NF-κB and IRAK4 and narrowed down the target(s) to the myddosome complex comprised of TLR4, MyD88 and MyD88 adaptor like protein (MAL). Preliminary data strongly indicate the glutathionylation of MAL on LPS stimulation which is abolished in GSTO1-1 deficient macrophages. The functional implications of the glutathionylation state of MAL are yet to be fully understood. Further studies are in progress to identify the underlying mechanism by which GSTO1-1 regulates TLR4 mediated inflammation in vivo.