A role for glutathione transferase omega 1 (GSTO1-1) in the glutathionylation cycle

Background: Glutathionylation is a major post-translational modification that regulates protein function. Results: Human glutathione transferase Omega 1 (GSTO1-1) can catalyze the deglutathionylation of protein thiols in vitro and in cell culture. Conclu

The biological roles of glutathione transferase Omega 1

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

GSTO1-1 plays a pro-inflammatory role in models of inflammation, colitis and obesity

Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo. In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.This work was supported by a grant from the Gretel and Gordon Bootes Medical Research Foundation to D.M. and P.B. The National Health and Medical Research Council of Australia (NHMRC) is thanked for Project Grant APP1124673 to PB, MC, LO, AO, and Fellowship support for J.B. (2012–2016 Senior Research Fellowship #1020411). J.B. acknowledges the Australian Federal Government Education Investment Fund Super Science Initiative and the Victorian State Government, Victoria Science Agenda Investment Fund for infrastructure support, and Translating Health Discovery (THD) NCRIS soft infrastructure support through Terapeutic Innovation Australia (TIA)

Human protein reference database—2006 update

Human Protein Reference Database (HPRD) () was developed to serve as a comprehensive collection of protein features, post-translational modifications (PTMs) and protein–protein interactions. Since the original report, this database has increased to >20 000 proteins entries and has become the largest database for literature-derived protein–protein interactions (>30 000) and PTMs (>8000) for human proteins. We have also introduced several new features in HPRD including: (i) protein isoforms, (ii) enhanced search options, (iii) linking of pathway annotations and (iv) integration of a novel browser, GenProt Viewer (), developed by us that allows integration of genomic and proteomic information. With the continued support and active participation by the biomedical community, we expect HPRD to become a unique source of curated information for the human proteome and spur biomedical discoveries based on integration of genomic, transcriptomic and proteomic data

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons

A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3- naphthalenedicarboxaldehyde

This study reports the development of a new assay for the rapid determination of protein glutathionylation in tissues and cell lines using commercially available reagents and standard instrumentation. In this method cells are homogenized in the presence of N-ethylmaleimide to eliminate free thiols and the proteins are precipitated with acetone. Subsequently, the disulfide-bound glutathione is eluted from the protein by the addition of tris(2-carboxyethyl)phosphine and reacted with 2,3-napthalenedicarboxaldehyde to generate a highly fluorescent product. Lymphoblastoid cell lines were found to have glutathionylation levels in the range of 0.3-3 nmol/mg protein, which were significantly elevated after treatment of the cells with S-nitrosoglutathione. Mouse tissues including liver, kidney, lung, heart, brain, spleen, and testes were found to have glutathionylation levels between 1 and 2.5 nmol/mg protein and the levels tended to increase after treatment of mice with doxorubicin. In contrast, mouse skeletal muscle glutathionylation was significantly higher (4.2 ± 0.33 nmol/mg, p < 0.001) than in other tissues in untreated mice and decreased to 1.9 ± 0.15 nmol/mg after doxorubicin treatment. This new method allows rapid measurement of cellular glutathionylation in a high-throughput 96-well plate format

Glutathione transferases, regulators of cellular metabolism and physiology

Background The cytosolic glutathione transferases (GSTs) comprise a super family of proteins that can be categorized into multiple classes with a mixture of highly specific and overlapping functions. Scope of review The review covers the genetics, struct

Structure, function and disease relevance of Omega-class glutathione transferases

The Omega-class cytosolic glutathione transferases (GSTs) have distinct structural and functional attributes that allow them to perform novel roles unrelated to the functions of other GSTs. Mammalian GSTO1-1 has been found to play a previously unappreciated role in the glutathionylation cycle that is emerging as significant mechanism regulating protein function. GSTO1-1-catalyzed glutathionylation or deglutathionylation of a key signaling protein may explain the requirement for catalytically active GSTO1-1 in LPS-stimulated pro-inflammatory signaling through the TLR4 receptor. The observation that ML175 a specific GSTO1-1 inhibitor can block LPS-stimulated inflammatory signaling has opened a new avenue for the development of novel anti-inflammatory drugs that could be useful in the treatment of toxic shock and other inflammatory disorders. The role of GSTO2-2 remains unclear. As a dehydroascorbate reductase, it could contribute to the maintenance of cellular redox balance and it is interesting to note that the GSTO2 N142D polymorphism has been associated with multiple diseases including Alzheimer’s disease, Parkinson’s disease, familial amyotrophic lateral sclerosis, chronic obstructive pulmonary disease, age-related cataract and breast cancer

Borderline ovarian mucinous tumor with anaplastic carcinomatous mural nodule: A case report

Malignant mural nodules in borderline ovarian tumors are rare. Among them, anaplastic mural nodules are infrequent and only limited case reports are available. Here we report a patient diagnosed as borderline ovarian mucinous tumor with an anaplastic carcinomatous mural nodule. She underwent comprehensive staging laparotomy and six cycles of adjuvant chemotherapy with paclitaxel – carboplatin and has no evidence of disease progression at eight months of follow up. This tumor has an aggressive behaviour and patients with stage ≥1C have inferior survival