Добірка наукової літератури з теми "GSTO1-1"

Omega class glutathione transferases, GSTO1-1 and GSTO2-2, exhibit different activities involved in regulation of inflammation, apoptosis and redox homeostasis. We investigated the the prognostic significance of GSTO1 (rs4925) and GSTO2 (rs156697 and rs2297235) polymorphisms in clear cell renal cell carcinoma (ccRCC) patients. GSTO1-1 and GSTO2-2 expression and phosphorylation status of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ /mammalian target of rapamycin (mTOR) and Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathways in non-tumor and tumor ccRCC tissue, as well as possible association of GSTO1-1 with signaling molecules were also assessed. GSTO genotyping was performed by quantitative PCR in 228 ccRCC patients, while expression and immunoprecipitation were analyzed by Western blot in 30 tissue specimens. Shorter survival in male carriers of GSTO1*C/C wild-type genotype compared to the carriers of at least one variant allele was demonstrated (p = 0.049). GSTO1*C/C genotype independently predicted higher risk of overall mortality among male ccRCC patients (p = 0.037). Increased expression of GSTO1-1 and GSTO2-2 was demonstrated in tumor compared to corresponding non-tumor tissue (p = 0.002, p = 0.007, respectively), while GSTO1 expression was correlated with interleukin-1β (IL-1β)/pro-interleukin-1β (pro-IL-1β) ratio (r = 0.260, p = 0.350). Interaction of GSTO1 with downstream effectors of investigated pathways was shown in ccRCC tumor tissue. This study demonstrated significant prognostic role of GSTO1 polymorphism in ccRCC. Up-regulated GSTO1-1 and GSTO2-2 in tumor tissue might contribute to aberrant ccRCC redox homeostasis.

Omega class glutathione transferase (GSTO) has been recently described in a number of mammalian species. We used immunohistochemistry to determine the cellular and tissue distribution of GSTO1–1 in humans. Expression of GSTO1–1 was abundant in a wide range of normal tissues, particularly liver, macrophages, glial cells, and endocrine cells. We also found nuclear staining in several types of cells, including glial cells, myoepithelial cells of the breast, neuroendocrine cells of colon, fetal myocytes, hepatocytes, biliary epithelium, ductal epithelium of the pancreas, Hoffbauer cells of the placenta, and follicular and C-cells of the thyroid. These observations and the known activity of GSTO1–1 suggest biological functions that are not shared with other GSTs. (J Histochem Cytochem 49:983–987, 2001)

Abstract Inflammatory bowel disease (IBD) is characterized by multiple alterations in cytokine expression and is a risk factor for colon cancer. The Omega class glutathione transferase GSTO1-1 regulates the release of the pro-inflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18) by deglutathionylating NEK7 in the NLRP3 inflammasome. When treated with azoxymethane and dextran sodium sulphate (AOM/DSS) as a model of IBD, Gsto1−/− mice were highly sensitive to colitis and showed a significant increase in the size and number of colon tumours compared with wild-type (WT) mice. Gsto1−/− mice treated with AOM/DSS had significantly lower serum IL-1β and IL-18 levels as well as significantly decreased interferon (IFN)-γ, decreased pSTAT1 and increased pSTAT3 levels in the distal colon compared with similarly treated WT mice. Histologically, AOM/DSS treated Gsto1−/− mice showed increased active chronic inflammation with macrophage infiltration, epithelial dysplasia and invasive adenocarcinoma compared with AOM/DSS treated WT mice. Thus, this study shows that GSTO1-1 regulates IL-1β and IL-18 activation and protects against colorectal cancer formation in the AOM/DSS model of IBD. The data suggest that while GSTO1-1 is a new target for the regulation of the NLRP3 inflammasome-associated cytokines IL-1β and IL-18 by small molecule inhibitors, there is a possibility that anti-inflammatory drugs targeting these cytokines may potentiate colon cancer in some situations.

Recent years have seen a growing evidence of ethnic differences in the frequency of glutathione S-transferase omega 1 (GSTO1) A140D gene polymorphism, which is associated with various cancers such as breast and liver. Until now however, no association has been investigated between the GSTO1 A140D polymorphism and lung cancer. The aim of our study was to see if there was one in the Turkish population. To do that, we identified GSTO1 A140D polymorphism in 214 unrelated healthy individuals and 172 patients with non-small cell lung cancer (NSCLC) using the polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) method. The frequencies of A/A (wild type), A/D (heterozygous mutant), and D/D (homozygous mutant) GSTO1 A140D genotypes in healthy subjects were 48 %, 41 %, and 11 %, respectively. In NSCLC patients they were 48 %, 45 %, and 7 %, respectively. We found no significant association between the GSTO1 A140D gene polymorphism and NSCLC or its histological subtypes, namely squamous cell carcinoma or adenocarcinoma. Furthermore, this polymorphism did not correlate with smoking. Our study is the first to show that the frequency of GSTO1 A140D gene polymorphism in the Turkish population is similar to other Caucasian populations and that this polymorphism is not associated with susceptibility to NSCLC.

The trace element arsenic naturally presents in the environment. Arsenic is the essential factor to the human body at low level, however it causes environmental pollution and have negative effects to health at high level. Recently, arsenic contamination as well as its effects on public health, especially infants and children is increasingly becoming important and serious issues in worldwide. Glutathione S-transferase omega-1 (GSTO1) is a phase II enzymatic detoxification of xenobiotics in variety of animals including humans; to catalyze the arsenic methylation. The difference of urinary arsenic component in each individual may relate to the genetic polymorphism. To evaluate the variations of single nucleotide polymorphisms of GSTO1, PCR-RFLP technology was ultilized. Single nucleotide polymorphisms (SNPs) genotype of 150 cohort blood samples at GSTO1 Thr->Asn (rs15032), GSTO1 Ala->Val (rs11509439) and GSTO1 Ala->Asp (rs4925) were detected. The association between GSTO1 polymorphisms and prenatal arsenic exposure was evaluated by statistical analysis such as SPSS software version 20, t-test and oneway ANOVA. The results showed that GSTO1 Ala->Asp (rs4925) was statistically associated with MMA/iAs (p = 0.041). Differences between ratio of MMA/iAs and genotypes were checked by Tukey-Kramer method, along with oneway ANOVA showed that Individuals taking the AA genotype had higher MMA/ iAs ratio than individuals carrying the CC genotype, with a statistically significant association (p = 0.044), also clearly higher than the individuals carrying AC genotype, significant at p = 0.046. Therefore, it is possible that individuals carrying the AA genotype in the polymorphism have higher arsenic excretion than individuals with CC and AC genotypes

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.

Analyses of protein sequences from diverse genomes have revealed the ubiquitous nature of multi-domain proteins. They form up to 70% of proteomes of most eukaryotic organisms. Yet, our understanding of protein structure, folding and evolution has been dominated by extensive studies on single-domain proteins. We provide quantitative treatment and proof for prevailing intuitive ideas on the strategies employed by nature to stabilize otherwise unstable domains. We find that domains incapable of independent stability are stabilized by favourable interactions with tethered domains in the multi-domain context. Natural variations (nsSNPs) at these sites alter communication between domains and affect stability leading to disease manifestation. We emphasize this by using explicit all-atom molecular dynamics simulations to study the interface nsSNPs of human Glutathione S-transferase omega 1. We show that domain-domain interface interactions constrain inter-domain geometry (IDG) which is evolutionarily well conserved. The inter-domain linkers modulate the interactions by varying their lengths, conformations and local structure, thereby affecting the overall IDG. These findings led to the development of a method to predict interfacial residues in multi-domain proteins based on difference evolutionary information extracted from at least two diverse domain architectures (single and multi-domain). Our predictions are highly accurate (∼85%) and specific (∼95%). Using predicted residues to constrain domain–domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. Further, we developed and employed an alignment-free approach based on local amino-acid fragment matching to compare sequences of multi-domain proteins. This is especially effective in the absence of proper alignments, which is usually the case for multi-domain proteins. Using this, we were able to recreate the existing Hanks and Hunter classification scheme for protein kinases. We also showed functional relationships among Immunoglobulin sequences. The clusters obtained were functionally distinct and also showed unique domain-architectures. Our analysis provides guidelines toward rational protein and interaction design which have attractive applications in obtaining stable fragments and domain constructs essential for structural studies by crystallography and NMR. These studies enable a deeper understanding of rapport of protein domains in the multi-domain context.