The temporal and spatial dynamics of glyoxalase I following excitoxicity and brain ischaemia

Abstract MG (methylglyoxal) is an inevitable metabolite derived from glycolysis leading to protein modification, mitochondrial dysfunction and cell death. The ubiquitous glyoxalase system detoxifies MG under GSH consumption by mean of Glo1 (glyoxalase I) as the rate-limiting enzyme. Neurons are highly vulnerable to MG, whereas astrocytes seem less susceptible due to their highly expressed glyoxalases. In neurodegenerative diseases, MG and Glo1 were found to be pivotal players in chronic CNS (central nervous system) diseases. Comparable results obtained upon MG treatment and NMDA (N-methyl-d-aspartate) receptor activation provided evidence of a possible link. Additional evidence was presented by alterations in Glo1 expression upon stimulation of excitotoxicity as an event in the aftermath of brain ischaemia. Glo1 expression was remarkably changed following ischaemia, and beneficial effects were found after exogenous application of Tat (transactivator of transcription)-Glo1. In summary, there are strong indications that Glo1 seems to be a suitable target to modulate the consequences of acute neuronal injury. The glyoxalase system and the relationship to diseases The glyoxalase system comprising Glo1 (glyoxalase I; EC 4.4.1.5) and Glo2 (glyoxalase II; EC 3.1.2.6) is the main detoxifying system of MG (methylglyoxal) within all mammalian cells MG reacts with reduced glutathione (GSH) to a hemithioacetal which is subsequently converted into S-Dlactoylgluthatione by the rate-limiting enzyme Glo1 and metabolized further to D-lactate by Glo2, restoring the used GSH. Glo1 is a zinc metalloenzyme with a molecular mass of 42 kDa consisting of two similar monomers with a molecular mass of 21 kDa In many studies, the cytotoxic effects of enhanced MG levels such as DNA breakage, cytokine production/release, ROS (reactive oxygen species) production, mitochondrial dysfunction and related caspase activation, as well as protein glycation and associated functional impairment, have been found Regarding these effects, changes in MG and Glo1 indicate a possible role in the development of diseases. Actually, Glo1 alterations are documented for diabetes, aging, tumorigenesis, multidrug-resistance and nephropath

Posttranslational Modification of Human Glyoxalase 1 Indicates Redox-Dependent Regulation

Background: Glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) are ubiquitously expressed cytosolic enzymes that catalyze the conversion of toxic a-oxo-aldehydes into the corresponding a-hydroxy acids using L-glutathione (GSH) as a cofactor. Human Glo1 exists in various isoforms; however, the nature of its modifications and their distinct functional assignment is mostly unknown. Methodology/Principal Findings: We characterized native Glo1 purified from human erythrocytes by mass spectrometry. The enzyme was found to undergo four so far unidentified posttranslational modifications: (i) removal of the N-terminal methionine 1, (ii) N-terminal acetylation at alanine 2, (iii) a vicinal disulfide bridge between cysteine residues 19 and 20, and (iv) a mixed disulfide with glutathione on cysteine 139. Glutathionylation of Glo1 was confirmed by immunological methods. Both, N-acetylation and the oxidation state of Cys 19/20, did not impact enzyme activity. In contrast, glutathionylation strongly inhibited Glo1 activity in vitro. The discussed mechanism for enzyme inhibition by glutathionylation was validated by molecular dynamics simulation. Conclusion/Significance: It is shown for the first time that Glo1 activity directly can be regulated by an oxidative posttranslational modification that was found in the native enzyme, i.e., glutathionylation. Inhibition of Glo1 by chemical reaction with its co-factor and the role of its intramolecular disulfides are expected to be important factors within th

Ethyl pyruvate combats human leukemia cells but spares normal blood cells

Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3β. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors

Ethyl pyruvate

The microbiota has a strong influence on health and disease in humans. A causative shift favoring pathobionts is strongly linked to diseases. Therefore, anti-microbial agents selectively targeting potential pathogens as well as their biofilms are urgently demanded. Here we demonstrate the impact of ethyl pyruvate, so far known as ROS scavenger and antiinflammatory agent, on planktonic microbes and biofilms. Ethyl pyruvate combats preferably the growth of pathobionts belonging to bacteria and fungi independent of the genera and prevailing drug resistance. Surprisingly, this anti-microbial agent preserves symbionts like Lactobacillus species. Moreover, ethyl pyruvate prevents the formation of biofilms and promotes matured biofilms dissolution. This potentially new anti-microbial and anti-biofilm agent could have a tremendous positive impact on human, veterinary medicine and technical industry as well

Modulation of GLO1 expression affects malignant properties of cells

The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect) characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO). Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1) that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed

第909回千葉医学会例会・第29回肺癌研究施設例会

The microbiota has a strong influence on health and disease in humans. A causative shift favoring pathobionts is strongly linked to diseases. Therefore, anti-microbial agents selectively targeting potential pathogens as well as their biofilms are urgently demanded. Here we demonstrate the impact of ethyl pyruvate, so far known as ROS scavenger and anti-inflammatory agent, on planktonic microbes and biofilms. Ethyl pyruvate combats preferably the growth of pathobionts belonging to bacteria and fungi independent of the genera and prevailing drug resistance. Surprisingly, this anti-microbial agent preserves symbionts like Lactobacillus species. Moreover, ethyl pyruvate prevents the formation of biofilms and promotes matured biofilms dissolution. This potentially new anti-microbial and anti-biofilm agent could have a tremendous positive impact on human, veterinary medicine and technical industry as well

Interaction of murine monoclonal subunit-specific antibodies with phosphofructokinase-1 from Saccharomyces cerevisiae

AbstractMurine monoclonal subunit-specific antibodies against heterooctameric phosphofructokinase-1 from Saccharomyces cerevisiae exhibiting no cross-reactivity were purified and characterized regarding complex formation with the native enzyme, immunological reactivity to the SDS-denatured subunits originating from native and proteolytically truncated phosphofructokinase, and protection against proteolytic degradation. Strong complex formation was found with one α-specific antibody. Western blotting employing different enzyme forms allowed to localize epitope sites of the α-polypeptide chain. Monoclonal antibodies protect phosphofructokinase-1 against chymotryptic degradation