Glyoxalase 1 Expression Research Articles

Stable Isotope Metabolic Labeling with a Novel 15N-Enriched Bacteria Diet for Improved Proteomic Analyses of Mouse Models for Psychopathologies

The identification of differentially regulated proteins in animal models of psychiatric diseases is essential for a comprehensive analysis of associated psychopathological processes. Mass spectrometry is the most relevant method for analyzing differences in protein expression of tissue and body fluid proteomes. However, standardization of sample handling and sample-to-sample variability are problematic. Stable isotope metabolic labeling of a proteome represents the gold standard for quantitative mass spectrometry analysis. The simultaneous processing of a mixture of labeled and unlabeled samples allows a sensitive and accurate comparative analysis between the respective proteomes. Here, we describe a cost-effective feeding protocol based on a newly developed 15N bacteria diet based on Ralstonia eutropha protein, which was applied to a mouse model for trait anxiety. Tissue from 15N-labeled vs. 14N-unlabeled mice was examined by mass spectrometry and differences in the expression of glyoxalase-1 (GLO1) and histidine triad nucleotide binding protein 2 (Hint2) proteins were correlated with the animals' psychopathological behaviors for methodological validation and proof of concept, respectively. Additionally, phenotyping unraveled an antidepressant-like effect of the incorporation of the stable isotope 15N into the proteome of highly anxious mice. This novel phenomenon is of considerable relevance to the metabolic labeling method and could provide an opportunity for the discovery of candidate proteins involved in depression-like behavior. The newly developed 15N bacteria diet provides researchers a novel tool to discover disease-relevant protein expression differences in mouse models using quantitative mass spectrometry.

Troglitazone reverses the multiple drug resistance phenotype in cancer cells

A major problem in treating cancer is the development of drug resistance. We previously demonstrated doxorubicin (DOX) resistance in K562 human leukemia cells that was associated with upregulation of glyoxalase 1 (GLO-1) and histone H3 expression. The thiazolidinedione troglitazone (TRG) downregulated GLO-1 expression and further upregulated histone H3 expression and post-translational modifications in these cells, leading to a regained sensitivity to DOX. Given the pleiotropic effects of epigenetic changes in cancer development, we hypothesized that TRG may downregulate the multiple drug resistance (MDR) phenotype in a variety of cancer cells. To test this, MCF7 human breast cancer cells and K562 cells were cultured in the presence of low-dose DOX to establish DOX-resistant cell lines (K562/DOX and MCF7/DOX). The MDR phenotype was confirmed by Western blot analysis of the 170 kDa P-glycoprotein (Pgp) drug efflux pump multiple drug resistance protein 1 (MDR-1), and the breast cancer resistance protein (BCRP). TRG markedly decreased expression of both MDR-1 and BCRP in these cells, resulting in sensitivity to DOX. Silencing of MDR-1 expression also sensitized MCF7/DOX cells to DOX. Use of the specific and irreversible peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor GW9662 in the nanomolar range not only demonstrated that the action of TRG on MCF/DOX was PPARgamma-independent, but indicated that PPARgamma may play a role in the MDR phenotype, which is antagonized by TRG. We conclude that TRG is potentially a useful adjunct therapy in chemoresistant cancers.

Glyoxalase‐1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C. elegans: progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C. elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C. elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C. elegans lifespan.

The characterisation and functional analysis of the human glyoxalase-1 gene using methods of bioinformatics

Methylglyoxal (MG), which forms MG-derived AGE, is elevated in diabetic subjects with vascular disease. Detoxification of MG occurs through the glyoxalase system incorporating glyoxalase-1 (GLO1) and glyoxalase-2. Perturbations of the glyoxalase-1 gene ( GLO1) may result in vulnerability to vascular complications through alterations in AGE interactions. We used bioinformatics to predict the structure, function and genetic variation of GLO1. We identified a previously unreported exon. Seventy single nucleotide polymorphisms (SNPs) were identified bioinformatically. The amino acid substitution Ala 111 Glu was confirmed and predicted to be tolerant. Though no alternative splice variants were identified, novel multiple alternative transcription start sites and alternative 3′ UTRs were demonstrated. Ubiquitous expression of GLO1 was confirmed. Conserved regulatory regions were predicted 5′ to the transcription start site and in the distal promoter, and several predicted conserved transcription regulatory elements were suggested in the 5′ UTR. This study of GLO1 demonstrates multiple sequence variants at DNA and mRNA levels, areas of sequence conservation and SNPs that are predicted to affect function. A differential ability of glyoxalase-1 to reduce the formation and subsequent interaction of AGEs may have a role in the structural and functional manifestations of diabetic vascular disease.