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Mitochondrial and synaptic dysfunction are pathological features of brain ageing and cognitive decline. Synaptic mitochondria are vital for meeting the high energy demands of synaptic transmission. However, little is known about the link between age-related metabolic changes and the integrity of synaptic mitochondria. To this end, we investigated the mechanisms of advanced glycation end product (AGE)-mediated mitochondrial and synaptic stress and evaluated the strategies to eliminate these toxic metabolites. Using aged brain and novel transgenic mice overexpressing neuronal glyoxalase 1 (GLO1), we comprehensively analysed alterations in accumulation/build-up of AGEs and related metabolites in synaptic mitochondria and the association of AGE levels with mitochondrial function. We demonstrated for the first time that synaptic mitochondria are an early and major target of AGEs and the related toxic metabolite methylglyoxal (MG), a precursor of AGEs. MG/AGE-insulted synaptic mitochondria exhibit deterioration of mitochondrial and synaptic function. Such accumulation of MG/AGEs positively correlated with mitochondrial perturbation and oxidative stress in ageing brain. Importantly, clearance of AGE-related metabolites by enhancing neuronal GLO1, a key enzyme for detoxification of AGEs, reduces synaptic mitochondrial AGE accumulation and improves mitochondrial and cognitive function in ageing and AGE-challenged mice. Furthermore, we evaluated the direct effect of AGEs on synaptic function in hippocampal neurons in live brain slices as an ex vivo model and in vitro cultured hippocampal neurons by recording long-term potentiation (LTP) and measuring spontaneously occurring miniature excitatory postsynaptic currents (mEPSCs). Neuronal GLO1 rescues deficits in AGE-induced synaptic plasticity and transmission by full recovery of decline in LTP or frequency of mEPSC. These studies explored crosstalk between synaptic mitochondrial dysfunction and age-related metabolic changes relevant to brain ageing and cognitive decline. Synaptic mitochondria are particularly susceptible to AGE-induced damage, highlighting the central importance of synaptic mitochondrial dysfunction in synaptic degeneration in age-related cognitive decline. Thus, augmenting GLO1 function to scavenge toxic metabolites represents a therapeutic approach to reduce age-related AGE accumulation and improve mitochondrial function and learning and memory. [ABSTRACT FROM AUTHOR]

Glyoxalase 1 (Glo I), a crucial enzyme with multifaceted roles in various physiological and pathological processes, has garnered significant attention in recent years. As a critical detoxification enzyme, Glo I plays a vital role in neutralizing methylglyoxal (MG), a highly reactive and toxic dicarbonyl compound that can irreversibly modify proteins, lipids, and nucleic acids, leading to the formation of advanced glycation end-products (AGEs). The assay involves incubating the Glo I enzyme with glutathione and methylglyoxal, forming S-D-lactoylglutathione. This compound is then converted to lactic acid by adding sulfuric acid, which is oxidized to acetaldehyde using copper. Finally, acetaldehyde reacts with p-phenylphenol in sulfuric acid, creating a colored complex that absorbs light at 570 nm. The study also rigorously examines potential interferences from various biochemical substances, demonstrating the robustness of the proposed method. Experiments were conducted using different combinations of interfering biomolecules, including monosaccharides, amino acids, and proteins, to evaluate their impact on Glo I activity. The results indicate that the p-phenylphenol-Glo I method maintains its effectiveness in the presence of these substances. Furthermore, the method's accuracy was rigorously assessed through intra-day and inter-day analyses, with results expressed as percentage relative error (RE% < 0.4%) to ensure precision in Glo I activity measurements. The current method exhibited excellent linearity across a broad range of 0.005-100 µM Lactate, with a high Pearson's correlation coefficient of 0.989. This work contributes valuable insights into the quantification of Glo I and holds significant implications for research in metabolic disorders and related fields. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MG), a highly reactive and cytotoxic α-oxoaldehyde compound, can over-accumulate under abiotic stress, consequently injuring plants or even causing death. Glyoxalase I (GLYI), the first enzyme of the glyoxalase pathway, plays multiple roles in the detoxification of MG and in abiotic stress responses. However, the GLY1 gene in maize has been little studied in response to abiotic stress. In this study, we screened a glyoxalase I gene (ZmGLYI-8) and overexpressed in Arabidopsis. This gene was localized in the cytoplasm and can be induced in maize seedlings under multiple stress treatments, including salt, drought, MG, ABA, H2O2 and high temperature stress. Phenotypic analysis revealed that after MG, salt and drought stress treatments, overexpression of ZmGLYI-8 increased the tolerance of transgenic Arabidopsis to MG, salt and drought stress. Furthermore, we demonstrated that the overexpression of ZmGLYI-8 scavenges accumulated reactive oxygen species, detoxifies MG and enhances the activity of antioxidant enzymes to improve the resistance of transgenic Arabidopsis plants to salt and drought stress. In summary, this study preliminarily elucidates the molecular mechanism of the maize ZmGLYI-8 gene in transgenic Arabidopsis and provides new insight into the breeding of salt- and drought-tolerant maize varieties. [ABSTRACT FROM AUTHOR]

In previous study we characterized the oncogenic role of long non-coding RNA MALAT1 in esophageal squamous cell carcinoma (ESCC), but the detailed mechanism remains obscure. Here we identified glyoxalase 1 (GLO1) as the most possible executor of MALAT1 by microarray screening. GLO1 is responsible for degradation of cytotoxic methylglyoxal (MGO), which is by-product of tumor glycolysis. Accumulated MGO may lead to glycation of DNA and protein, resulting in elevated advanced glycation end products (AGEs), while glyoxalase 1 detoxify MGO to alleviate its cytotoxic effect to tumor cells. GLO1 interfering led to accumulation of AGEs and following activation of DNA injury biomarkers, which lead to cell cycle arrest and growth inhibition. In silico analysis based on online database revealed abundant enrichment of histone acetylation marker H3K27ac in GLO1 promotor, and acetyltransferase inhibitor C646 declined GLO1 expression. Acetyltransferase KAT2B, which was also identified as a target of MALAT, mediated histone lysine acetylation of GLO1 promotor, which was confirmed by ChIP-qPCR experiment. Shared binding sites of miR-206 were found on MALAT1 and KAT2B mRNA. Dual-luciferase reporter assays confirmed interaction within MALAT1-miR-206-GLO1. Finally, we identified MALAT1 encapsuled by exosome from donor cells, and transferred malignant behaviors to recipient cells. The secreted exosomes may enter circulation, and serum MALAT1 level combined with traditional tumor markers showed potential power for ESCC diagnosis.

Glyoxalase I (GLO I), a major enzyme involved in the detoxification of the anaerobic glycolytic byproduct methylglyoxal, is highly expressed in various tumors, and is regarded as a promising target for cancer therapy. We recently reported that piceatannol potently inhibits human GLO I and induces the death of GLO I‐dependent cancer cells. Pyruvate kinase M2 (PKM2) is also a potential therapeutic target for cancer treatment, so we evaluated the combined anticancer efficacy of piceatannol plus low‐dose shikonin, a potent and specific plant‐derived PKM2 inhibitor, in two GLO I‐dependent cancer cell lines, HL‐60 human myeloid leukemia cells and NCI‐H522 human non‐small‐cell lung cancer cells. Combined treatment with piceatannol and low‐dose shikonin for 48 h synergistically reduced cell viability, enhanced apoptosis rate, and increased extracellular methylglyoxal accumulation compared to single‐agent treatment, but did not alter PKM1, PKM2, or GLO I protein expression. Taken together, these results indicate that concomitant use of low‐dose shikonin potentiates piceatannol‐induced apoptosis of GLO I‐dependent cancer cells by augmenting methylglyoxal accumulation. [ABSTRACT FROM AUTHOR]

Oxidative and dicarbonyl stress, driven by excess accumulation of glycolytic intermediates in cells that are highly permeable to glucose in the absence of effective insulin activity, appear to be the chief mediators of the complications of diabetes. The most pathogenically significant dicarbonyl stress reflects spontaneous dephosphorylation of glycolytic triose phosphates, giving rise to highly reactive methylglyoxal. This compound can be converted to harmless lactate by the sequential activity of glyoxalase I and II, employing glutathione as a catalyst. The transcription of glyoxalase I, rate-limiting for this process, is promoted by Nrf2, which can be activated by nutraceutical phase 2 inducers such as lipoic acid and sulforaphane. In cells exposed to hyperglycemia, glycine somehow up-regulates Nrf2 activity. Zinc can likewise promote glyoxalase I transcription, via activation of the metal-responsive transcription factor (MTF) that binds to the glyoxalase promoter. Induction of glyoxalase I and metallothionein may explain the protective impact of zinc in rodent models of diabetic complications. With respect to the contribution of oxidative stress to diabetic complications, promoters of mitophagy and mitochondrial biogenesis, UCP2 inducers, inhibitors of NAPDH oxidase, recouplers of eNOS, glutathione precursors, membrane oxidant scavengers, Nrf2 activators, and correction of diabetic thiamine deficiency should help to quell this.

The health-promoting properties of natural plant bioactive compounds are mainly attributable to their ability to counteract oxidative stress. This is considered a major causative factor in aging and aging-related human diseases, in which a causal role is also ascribed to dicarbonyl stress. This is due to accumulation of methylglyoxal (MG) and other reactive dicarbonyl species, leading to macromolecule glycation and cell/tissue dysfunction. The glyoxalase (GLYI) enzyme, catalyzing the rate-limiting step of the GSH-dependent MG detoxification pathway, plays a key role in cell defense against dicarbonyl stress. Therefore, the study of GLYI regulation is of relevant interest. In particular, GLYI inducers are important for pharmacological interventions to sustain healthy aging and to improve dicarbonyl-related diseases; GLYI inhibitors, allowing increased MG levels to act as proapoptotic agents in tumor cells, are of special interest in cancer treatment. In this study, we performed a new in vitro exploration of biological activity of plant bioactive compounds by associating the measurement of their antioxidant capacity (AC) with the evaluation of their potential impact on dicarbonyl stress measured as capability to modulate GLYI activity. AC was evaluated using TEAC, ORAC, and LOX-FL methods. The GLYI assay was performed using a human recombinant isoform, in comparison with the recently characterized GLYI activity of durum wheat mitochondria. Different plant extracts were tested, obtained from plant sources with very high phytochemical content ('Sun Black' and wildtype tomatoes, black and 'Polignano' carrots, and durum wheat grain). Results showed high antioxidant properties of the tested extracts, associated with different modes (no effect, activation, and inhibition) and effectiveness in modulating both GLYI activity sources. Overall, results indicate the GLYI assay as an advisable and promising tool for researching plant foods as a source of natural antioxidant compounds acting as GLYI enzymatic regulators to be used for dietary management associated the treatment of oxidative/dicarbonyl-promoted diseases. [ABSTRACT FROM AUTHOR]

Suaad Abdallah Audat,1 Qosay Ali Al-Balas,2 Buthina Abdallah Al-Oudat,2 Mo’ad Jamil Athamneh,1 Amanda Bryant-Friedrich3 1Department of Chemistry, College of Science and Arts, Jordan University of Science and Technology, Irbid, 22110, Jordan; 2Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, 22110, Jordan; 3Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, 48202, USACorrespondence: Suaad Abdallah Audat, Department of Chemistry, College of Science and Arts, Jordan University of Science and Technology, P.O. Box 3030, Irbid, 22110, Jordan, Tel +962 2 720 1000, Fax +962 2 720 1071, Email saaudat@just.edu.joBackground: Glyoxalase system is one of the defense cellular mechanisms that protect cells against endogenous harmful metabolites, mainly methylglyoxal (MG), through conversion of cytotoxic methylglyoxal into the non-toxic lactic acid. Glyoxalase system comprises of two enzymes glyoxalase I, glyoxalase II, and a catalytic amount of reduced glutathione. Cancerous cells overexpress glyoxalase I, making it a target for cancer therapy. Many studies have been conducted to identify potent Glx-I inhibitors.Methods: Aiming to discover and develop novel Glx-I inhibitors, a series of 1,4-benzenesulfonamide derivatives were designed, synthesized, and biologically evaluated in vitro against human Glx-I enzyme. Seventeen compounds were designed based on the hit compound that was obtained from searching the National Cancer Institute (NCI) database. The synthesis of the target compounds ( 13– 29) was accomplished utilizing an azo coupling reaction of aniline derivatives and activated substituted aromatic compounds. To understand the binding mode of the active compounds at the active site of Glx-I, docking studies were performed.Results: Structure activity relationship (SAR) studies were accomplished which led to the identification of several compounds that showed potent inhibitory activity with IC50 values below 10 μM. Among the compounds tested, compounds (E)-2-hydroxy-5-((4-sulfamoylphenyl)diazenyl)benzoic acid ( 26) and (E)-4-((8-hydroxyquinolin-5-yl)diazenyl) benzenesulfonamide ( 28) displayed potent Glx-I inhibitory activity with IC50 values of 0.39 μM and 1.36 μM, respectively. Docking studies of compounds 26 and 28 were carried out to illustrate the binding mode of the molecules into the Glx-I active site.Conclusion: Our results show that compounds 26 and 28 displayed potent Glx-I inhibitory activity and can bind the Glx-I well. These findings should lead us to discover new classes of compounds with better Glx-I inhibition.Graphical Abstract: Keywords: anticancer agents, glyoxalase I, structure activity relationship, SAR, molecular docking

Organobismuth compounds, i.e., organic-inorganic hybrid molecules composed of an organic structure and bismuth metal, have been reported to induce cytotoxicity in cancer cells; however, the target proteins associated with this cytotoxicity have not been elucidated. Herein, we investigated the inhibitory effect of five organobismuth compounds on human glyoxalase I (hGLO I), a promising target candidate for cancer therapy. Among these compounds, triphenylbismuth dichloride (Bi-05) exerted a strong inhibitory effect on hGLO I. Indeed, Bi-05 inhibited hGLO I in a dose-dependent manner with an IC50 value of 0.18 µM. Bi-05 also induced cytotoxicity in human leukemia HL-60 cells and human lung cancer NCI-H522 cells, both of which exhibit high expression levels of GLO I. However, the hGLO I-inhibiting and cytotoxic effects of Bi-05 disappeared when the bismuth atom was replaced with an antimony or phosphorus atom. Bismuth(III) nitrate had little inhibitory effect on hGLO I activity and only slightly reduced the viability of cancer cells. In the culture medium of Bi-05-treated HL-60 cells, the concentration of the GLO I substrate methylglyoxal was markedly elevated. In addition, Bi-05 treatment more strongly inhibited human lung cancer NCI-H522 cell (exhibiting high GLO I expression) proliferation than human lung cancer NCI-H460 cell (exhibiting low GLO I expression) proliferation. Furthermore, the cytotoxicity of Bi-05 was significantly decreased by pre- and co-treatment with the methylglyoxal scavengers N-acetyl-L-cysteine and aminoguanidine. Overall, these results suggest that Bi-05 treatment leads to the accumulation of methylglyoxal via GLO I inhibition, resulting in cytotoxic effects in cancer cells. [ABSTRACT FROM AUTHOR]

Background: Methylglyoxal (MGO) is a known toxic byproduct of glycolysis, with MGO-induced cytotoxicity believed to contribute to the pathogenesis of several diseases. Glyoxalase I (GLO1) is a key enzyme for eliminating MGO in mammalian cells, therefore, compounds affecting GLO1 activity are potential therapeutic agents for MGO-induced disorders. Previously, we found nordihydroguaiaretic acid (NDGA) as a potent GLO1 inhibitor. Methods: The inhibitory characteristics of NDGA were determined spectrophotometrically with recombinant GLO1. NDGA-induced growth-inhibition and accumulation of MGO-derived advanced glycation end products (AGEs) were examined in EA.hy926 cells. Results: NDGA showed significant inhibition of GLO1 enzymatic activity in a dose-dependent manner. Its Ki value was estimated to be 146-fold lower than that of myricetin, a known GLO1 inhibitor. The co-addition of MGO with NDGA to the cells resulted in significant growth inhibition, suggesting that MGO accumulation, sufficient to affect cell growth, was caused by NDGA inhibiting GLO1. These findings were supported by the observations that the addition of aminoguanidine, a typical MGO scavenger, significantly reversed cell-growth inhibition by co-addition of MGO with NDGA, and that an increase in intracellular MGO-derived AGEs was observed during incubation with the co-addition of MGO with NDGA. Conclusion: NDGA was found to be a novel and potent inhibitor of GLO1. The co-addition of NDGA with MGO to the cells resulted in increased intracellular MGO accumulation followed by enhanced cell-growth inhibition. [ABSTRACT FROM AUTHOR]

The glyoxalase pathway plays a vital role in the chemical detoxification of methylglyoxal (MG) in biological systems. Our previous study suggested that OsGLYI3 may be effective in seed natural aging. In this study, the rice OsGLYI3 gene was cloned and characterized as specifically expressed in the seed. The accelerated aging (AA) treatment results indicated significant roles of OsGLYI3 in seed longevity and vigor, as the seeds of the transgenic lines with overexpressed and knocked-out OsGLYI3 exhibited higher and lower germination, respectively. The AA treatment also increased the superoxide dismutase (SOD) activity in the overexpressed transgenic seeds compared to the wild-type seeds yet lowered the SOD activity in the CRISPR/Cas9-derived transgenic rice lines. Rice OsGLYI3 was markedly upregulated in response to NaCl induced stress conditions. Compared to wild-type plants, overexpressed transgenic rice lines exhibited increased GLYI activity, decreased MG levels and improved salt stress tolerance, while CRISPR/Cas9 knockout transgenic rice lines showed decreased glyoxalase I activity, increased MG levels, and greater sensitivity to stress treatments with NaCl. Collectively, our results confirmed for the first time that OsGLYI3 is specifically expressed in rice seeds and contributes to seed longevity and salt stress tolerance. • OsGLYI3 was confirmed for the first time specifically expressed in rice seed. • OsGLYI3 exhibits a detoxification function on methylglyoxal (MG). • OsGLYI3 plays an important role in rice seed longevity and vigor. • OsGLYI3 is essential for stress tolerance improvement in rice. [ABSTRACT FROM AUTHOR]

Glyoxalase I (GLYI) catalyzes the rate-limiting step of the glyoxalase pathway that, in the presence of GSH, detoxifies the cytotoxic molecule methylglyoxal (MG) into the non-toxic D-lactate. In plants, MG levels rise under various abiotic stresses, so GLYI may play a crucial role in providing stress tolerance. In this study, a comprehensive genome database analysis was performed in durum wheat (Triticum durum Desf.), identifying 27 candidate GLYI genes (TdGLYI). However, further analyses of phylogenetic relationships and conserved GLYI binding sites indicated that only nine genes encode for putative functionally active TdGLYI enzymes, whose distribution was predicted in three different subcellular compartments, namely cytoplasm, plastids and mitochondria. Expression profile by qRT-PCR analysis revealed that most of the putative active TdGLYI genes were up-regulated by salt and osmotic stress in roots and shoots from 4-day-old seedlings, although a different behavior was observed between the two types of stress and tissue. Accordingly, in the same tissues, hyperosmotic stress induced an increase (up to about 40%) of both GLYI activity and MG content as well as a decrease of GSH (up to about –60%) and an increase of GSSG content (up to about 7-fold) with a consequent strong decrease of the GSH/GSSG ratio (up to about –95%). Interestingly, in this study, we reported the first demonstration of the existence of GLYI activity in highly purified mitochondrial fraction. In particular, GLYI activity was measured in mitochondria from durum wheat (DWM), showing hyperbolic kinetics with Km and Vmax values equal to 92 ± 0.2 μM and 0.519 ± 0.004 μmol min–1 mg–1 of proteins, respectively. DWM–GLYI resulted inhibited in a competitive manner by GSH (Ki = 6.5 ± 0.7 mM), activated by Zn2+ and increased, up to about 35 and 55%, under salt and osmotic stress, respectively. In the whole, this study provides basis about the physiological significance of GLYI in durum wheat, by highlighting the role of this enzyme in the early response of seedlings to hyperosmotic stress. Finally, our results strongly suggest the existence of a complete mitochondrial GLYI pathway in durum wheat actively involved in MG detoxification under hyperosmotic stress. [ABSTRACT FROM AUTHOR]

SUMMARY: Methylglyoxal (MG) is a toxic by‐product of the glycolysis pathway in most living organisms and was previously shown to inhibit seed germination. MG is detoxified by glyoxalase I and II family proteins in plants. MG is abundantly produced during early embryogenesis in Arabidopsis seeds. However, the mechanism that alleviates the toxic effect of MG in maturing seeds is poorly understood. In this study, by T‐DNA mutant population screening, we found that mutations in a glyoxalase I gene (named GERMINATION‐IMPAIRED GLYOXALASE 1, GIG1) led to significantly impaired germination compared with wild‐type seeds. Transformation of full‐length GIG1 cDNA under the constitutively active cauliflower mosaic virus 35S promoter in the gig1 background completely recovered the seed germination phenotype. Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analyses revealed that GIG1 is uniquely expressed in seeds and is upregulated by abscisic acid (ABA) and downregulated by gibberellic acid (GA) during seed germination. An ABA signaling component, ABI3, directly activated GIG1 in maturing seeds. In addition, PHYTOCHROME INTERACTING FACTOR 1 (PIF1) also plays cooperatively with ABI3 in the regulation of GIG1 expression in the early stage of imbibed seeds. Furthermore, GIG1 expression is stably silenced by epigenetic repressors such as polycomb repressor complexes. Altogether, our results indicate that light and ABA signaling cooperate to enhance seed germination by the upregulation of GIG1 to detoxify MG in maturing seeds. Significance Statement: Two key transcriptional regulators, ABI3 and PIF1 act in a coordinated manner to detoxify methylglyoxal via activation of seed‐specific glyoxalase I gene named GIG1 and enhance seed germination in Arabidopsis plants. [ABSTRACT FROM AUTHOR]

Human glyoxalase I (hGLO I) is an enzyme for detoxification of methylglyoxal (MG) and has been considered an attractive target for the development of new anticancer drugs. In our previous report, the GLO I inhibitor TLSC702 induced apoptosis in tumor cells. Here, we determined the crystal structures of hGLO I and its complex with TLSC702. In the complex, the carboxyl O atom of TLSC702 is coordinated to Zn²+, and TLSC702 mainly shows van der Waals interaction with hydrophobic residues. In the inhibitor-unbound structure, glycerol, which has similar functional groups to MG, was bound to Zn2+, indicating that GLO I can easily bind to MG. This study provides a structural basis to develop better anticancer drugs. [ABSTRACT FROM AUTHOR]

Glyoxalase I (GLYI) catalyzes the rate-limiting step of the glyoxalase pathway that, in the presence of GSH, detoxifies the cytotoxic molecule methylglyoxal (MG) into the non-toxic D-lactate. In plants, MG levels rise under various abiotic stresses, so GLYI may play a crucial role in providing stress tolerance. In this study, a comprehensive genome database analysis was performed in durum wheat (Triticum durum Desf.), identifying 27 candidate GLYI genes (TdGLYI). However, further analyses of phylogenetic relationships and conserved GLYI binding sites indicated that only nine genes encode for putative functionally active TdGLYI enzymes, whose distribution was predicted in three different subcellular compartments, namely cytoplasm, plastids and mitochondria. Expression profile by qRT-PCR analysis revealed that most of the putative active TdGLYI genes were up-regulated by salt and osmotic stress in roots and shoots from 4-day-old seedlings, although a different behavior was observed between the two types of stress and tissue. Accordingly, in the same tissues, hyperosmotic stress induced an increase (up to about 40%) of both GLYI activity and MG content as well as a decrease of GSH (up to about –60%) and an increase of GSSG content (up to about 7-fold) with a consequent strong decrease of the GSH/GSSG ratio (up to about –95%). Interestingly, in this study, we reported the first demonstration of the existence of GLYI activity in highly purified mitochondrial fraction. In particular, GLYI activity was measured in mitochondria from durum wheat (DWM), showing hyperbolic kinetics with Km and Vmax values equal to 92 ± 0.2 μM and 0.519 ± 0.004 μmol min–1 mg–1 of proteins, respectively. DWM–GLYI resulted inhibited in a competitive manner by GSH (Ki = 6.5 ± 0.7 mM), activated by Zn2+ and increased, up to about 35 and 55%, under salt and osmotic stress, respectively. In the whole, this study provides basis about the physiological significance of GLYI in durum wheat, by highlighting the role of this enzyme in the early response of seedlings to hyperosmotic stress. Finally, our results strongly suggest the existence of a complete mitochondrial GLYI pathway in durum wheat actively involved in MG detoxification under hyperosmotic stress.

The health-promoting properties of natural plant bioactive compounds are mainly attributable to their ability to counteract oxidative stress. This is considered a major causative factor in aging and aging-related human diseases, in which a causal role is also ascribed to dicarbonyl stress. This is due to accumulation of methylglyoxal (MG) and other reactive dicarbonyl species, leading to macromolecule glycation and cell/tissue dysfunction. The glyoxalase (GLYI) enzyme, catalyzing the rate-limiting step of the GSH-dependent MG detoxification pathway, plays a key role in cell defense against dicarbonyl stress. Therefore, the study of GLYI regulation is of relevant interest. In particular, GLYI inducers are important for pharmacological interventions to sustain healthy aging and to improve dicarbonyl-related diseases; GLYI inhibitors, allowing increased MG levels to act as proapoptotic agents in tumor cells, are of special interest in cancer treatment. In this study, we performed a new in vitro exploration of biological activity of plant bioactive compounds by associating the measurement of their antioxidant capacity (AC) with the evaluation of their potential impact on dicarbonyl stress measured as capability to modulate GLYI activity. AC was evaluated using TEAC, ORAC, and LOX-FL methods. The GLYI assay was performed using a human recombinant isoform, in comparison with the recently characterized GLYI activity of durum wheat mitochondria. Different plant extracts were tested, obtained from plant sources with very high phytochemical content (‘Sun Black’ and wildtype tomatoes, black and ‘Polignano’ carrots, and durum wheat grain). Results showed high antioxidant properties of the tested extracts, associated with different modes (no effect, activation, and inhibition) and effectiveness in modulating both GLYI activity sources. Overall, results indicate the GLYI assay as an advisable and promising tool for researching plant foods as a source of natural antioxidant compounds acting as GLYI enzymatic regulators to be used for dietary management associated the treatment of oxidative/dicarbonyl-promoted diseases.

The sbiT-sbiR-sbiS operon of Stenotrophomonas maltophilia encodes an inner-membrane protein SbiT and a SbiS-SbiR two-component regulatory system. A sbiT mutant displayed a growth defect in LB agar. Mechanism studies revealed that sbiT deletion resulted in SbiSR activation and gloIo upregulation, which increased intracellular ROS level and caused growth defect., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Methylglyoxal (MG) is ubiquitously produced in all living organisms as a byproduct of glycolysis, higher levels of which are cytotoxic, leading to oxidative stress and apoptosis in the living systems. Though its generation is spontaneous but its detoxification involves glyoxalase pathway genes. Based on this understanding, the present study describes the possible role of MG as a novel non-antibiotic-based selection agent in rice. Further, by metabolizing MG, the glyoxalase pathway genes viz. glyoxalase I (GLYI) and glyoxalase II (GLYII), may serve as selection markers. Therefore, herein, transgenic rice harboring GLYI-GLYII genes (as selection markers) were developed and the effect of MG as a selection agent was assessed. The 3 mM MG concentration was observed as optimum for the selection of transformed calli, allowing efficient callus induction and proliferation along with high regeneration frequency (55 ± 2%) of the transgenic calli. Since the transformed calli exhibited constitutively higher activity of GLYI and GLYII enzymes compared to the wild type calli, the rise in MG levels was restricted even upon exogenous addition of MG during the selection process, resulting in efficient selection of the transformed calli. Therefore, MG-based selection method is a useful and efficient system for selection of transformed plants without significantly compromising the transformation efficiency. Further, this MG-based selection system is bio-safe and can pave way towards better public acceptance of transgenic plants. [ABSTRACT FROM AUTHOR]

Summary: Glyoxalase I (GLYI) is part of the glyoxalase system; its major function is the detoxification of α‐ketoaldehydes, including the potent and cytotoxic methylglyoxal (MG). Methylglyoxal disrupts mitochondrial respiration and increases production of reactive oxygen species (ROS), which also increase during pathogen infection of plant tissues; however, there have been few studies relating the glyoxalase system to the plant pathogen response. We used the promoter of VvGLYI‐4 to screen the upstream transcription factors and report a NAC (NAM/ATAF/CUC) domain‐containing transcription factor VvNAC72 in grapevine, which is localized to the nucleus. Our results show that VvNAC72 expression is induced by downy mildew, Plasmopara viticola, while the transcript level of VvGLYI‐4 decreases. Further analysis revealed that VvNAC72 can bind directly to the promoter region of VvGLYI‐4 via the CACGTG element, leading to inhibition of VvGLYI‐4 transcription. Stable overexpression of VvNAC72 in grapevine and tobacco showed a decreased expression level of VvGLYI‐4 and increased content of MG and ROS, as well as stronger resistance to pathogen stress. Taken together, these results demonstrate that grapevine VvNAC72 negatively modulates detoxification of MG through repression of VvGLYI‐4, and finally enhances resistance to downy mildew, at least in part, via the modulation of MG‐associated ROS homeostasis through a salicylic acid‐mediated defense pathway. [ABSTRACT FROM AUTHOR]

We investigated the effect of cyanidin-3-rutinoside (C3R) against methylglyoxal (MG)-advanced glycation end products (AGEs) mediated cardio-vascular abnormalities in rats pre-treated with MG. In the rats, plasma MG level rose following oral administration of MG (60–120 mg/rat/day) for 8 weeks and was accompanied by a buildup of MG-derived AGE, Nɛ-(carboxymethyl) lysine, in aortic tissue both of which were decreased by co-treatment with C3R (30 and 100 mg/kg/day). Similarly, MG-impaired vascular contraction/relaxation responses were normalized by C3R with concomitant upregulation of endothelial nitric oxide synthase mRNA expression. In the whole animal subjected to coronary artery ligation, C3R (100 mg/kg/day) reduced ventricular extra beats and ventricular tachycardia precipitated during acute ischemic episode. We suggest that C3R reduces the formation and accumulation of AGEs via direct trapping of the MG and upregulation of mRNA expression of glyoxalase I. The findings implicate C3R as a candidate compound for preventing MG-AGE mediated cardiovascular abnormalities in diabetes.

Members of the Brassicaceae family have the ability to regulate pollination events occurring on the stigma surface. In Brassica species, self-pollination leads to an allele-specific interaction between the pollen small cysteine-rich peptide ligand (SCR/SP11) and the stigmatic S-receptor kinase (SRK) that activates the E3 ubiquitin ligase ARC1 (Armadillo repeat-containing 1), resulting in proteasomal degradation of various compatibility factors including glyoxalase I (GLO1) which is necessary for successful pollination. In Brassica napus, the suppression of GLO1 was sufficient to reduce compatibility, and overexpression of GLO1 in self-incompatible Brassica napus stigmas resulted in partial breakdown of the self-incompatibility response. Here, we verified if BnGLO1 could function as a compatibility factor in the artificial self-incompatibility system of Arabidopsis thaliana expressing AlSCRb, AlSRKb and AlARC1 proteins from A. lyrata. Overexpression of BnGLO1 is sufficient to breakdown self-incompatibility response in A. thaliana stigmas. Therefore, GLO1 has an indisputable role as a compatibility factor in the stigma in regulating pollen attachment and pollen tube growth. Lastly, this study demonstrates the usefulness of an artificial self-incompatibility system in A. thaliana for interspecific self-incompatibility studies. [ABSTRACT FROM AUTHOR]

The accumulation of a metabolic by product – methylglyoxal above a minimal range can be highly toxic in all organisms. Stress induced elevation in methylglyoxal inactivates proteins and nucleic acids. Glutathione dependent glyoxalase enzymes like glyoxalase I and glyoxalase II together with glutathione independent glyoxalase III play inevitable role in methylglyoxal detoxification. Glyoxalase genes are generally conserved but with obvious exceptions. Mangroves being potent harsh land inhabitants, their internal organelles are constantly been exposed to elevated levels of methylglyoxal. First and foremost it is important to detect the presence of glyoxalases in mangroves. De novo transcriptome analysis of mangrove species Rhizophora mucronata Lam., identified eleven putative glyoxalase proteins (RmGLYI-1 to 5, RmGLYII-1 to 5 and RmGLYIII). Molecular characterization proposed PLN02300 or PLN02367 as the key domains of RmGLYI proteins. They possess molecular weight ranging from 26.45 to 32.53 kDa and may localize in cytosol or chloroplast. RmGLYII proteins of molecular weight 28.64–36 kDa, carrying PLN02398 or PLN02469 domains are expected to be localized in diverse cellular compartments. Cytosolic RmGLYIII with DJ-1/PfpI domain carries a molecular weight 26.4 kDa. Detailed structural analysis revealed monomeric nature of RmGLYI-1 and RmGLYII-1 whereas RmGLYIII is found to be homodimer. Molecular phylogenetic analysis and multiple sequence alignment specified conserved metal ion/substrate binding residues of RmGLY proteins. Estimation of relative expression of glyoxalases under salt stress indicated the prominence of RmGLYI and RmGLYII over RmGLYIII. The aforementioned prominence is supported by salt induced expression difference of glutathione metabolic enzymes and glutathione regulated transporter protein. • Reference sequence and annotation datasets from the de novo transcriptome of salt tolerant mangrove, Rhizophora mucronata. • GSH dependent and independent glyoxalase system in extremophilic mangroves. • Structural/biochemical characteristics of mangrove glyoxalase proteins. • Stability of glyoxalase structure models under salt stress; a lead for unidentified stability aspects of mangrove proteins. • Salt induced gene expression of mangrove glyoxalases correlated to GSH metabolic enzymes and GSH regulated transporter. [ABSTRACT FROM AUTHOR]

The detoxification of cadmium (Cd) or chromium (Cr) by microorganisms plays a vital role in bacterial survival and restoration of the polluted environment, but how microorganisms detoxify Cd and Cr simultaneously is largely unknown. Here, we isolated a bacterium, Cupriavidus sp. MP-37, which immobilized Cd(II) and reduced Cr(VI) simultaneously. Notably, strain MP-37 exhibited variable Cd(II) immobilization phenotypes, namely, cell adsorption and extracellular immobilization in the co-presence of Cd(II) and Cr(VI), while cell adsorption in the presence of Cd(II) alone. To unravel Cr(VI)-induced extracellular Cd(II) immobilization, proteomic analysis was performed, and methylglyoxal-scavenging protein (glyoxalase I, GlyI) and a regulator (YafY) showed the highest upregulation in the co-presence of Cd(II) and Cr(VI). GlyI overexpression reduced the intracellular methylglyoxal content and increased the immobilized Cd(II) content in extracellular secreta. The addition of lactate produced by GlyI protein with methylglyoxal as substrate increased the Cd(II) content in extracellular secreta. Reporter gene assay, electrophoretic mobility shift assay, and fluorescence quenching assay demonstrated that glyI expression was induced by Cr(VI) but not by Cd(II), and that YafY positively regulated glyI expression by binding Cr(VI). In the pot experiment, inoculation with the MP-37 strain reduced the Cd content of Oryza sativa L., and their secreted lactate reduced the Cr accumulation in Oryza sativa L. This study reveals that Cr(VI)-induced detoxification system drives methylglyoxal scavenging and Cd(II) extracellular detoxification in Cd(II) and Cr(VI) co-existence environment. [Display omitted] • Strain MP-37 displays simultaneous Cd(II) removal and Cr(VI) reduction abilities. • A Cr(VI)-induced methylglyoxal detoxification system increased Cd(II) removal. • Yafy and GlyI are key factors in the methylglyoxal detoxification system. • YafY positively regulated glyI expression, and GlyI was a Cr(VI)-binding protein. • The variable detoxification strategy of Cd(II) by bacteria favors survival. [ABSTRACT FROM AUTHOR]

• Biosensor for the detection of diabetes biomarker 'methylglyoxal' in blood plasma. • Sensitivity of 1.02 mA µM−1 and LOD of 0.21 µM were observed. • Biosensor responses for 350 human blood plasma samples were recorded. • Cross-validation of the biosensor with ELISA technique showed a 90% of correlation. Methylglyoxal (MG), a highly reactive by-product of glycolysis, is involved in the formation of advanced glycation end-products (AGEs). Elevated levels of MG have been correlated with micro-and macro-angiopathic complications in diabetes, including neuropathy, kidney disease, retinopathy, and cardiovascular disease. Therefore, point-of-care devices for detecting MG may be of great use in the screening of diabetes complications. This study was designed to determine the utility of the developed electrochemical biosensor to measure the level of MG in human plasma from type-2 diabetes mellitus patients. Electrochemical studies were carried out with optimized experimental parameters using the modified Platinum-electrode. Subsequently, clinical studies using 350 blood plasma samples were conducted and the results were validated against the ELISA kit, Normal Glucose Tolerance (NGT), and glycosylated haemoglobin (HbA1c). The MG sensor exhibited a linear range of 1.0–7.5 μM concentration with a sensitivity of 1.02 mA µM−1, a limit of detection of 0.21 µM, a limit of quantification of 0.70 µM and a response time less than 10 s. The sensor showed 90% correlation with ELISA data. The developed biosensor showed a significant correlation with HbA1c and fasting plasma glucose suggesting that it can be used as a point-of-care device to screen for diabetes. [ABSTRACT FROM AUTHOR]

Methylglyoxal is a common cytotoxic metabolite produced in plants during multiple biotic and abiotic stress. To mitigate the toxicity of MG, plants utilize the glyoxalase pathway comprising glyoxalase I (GLYI), glyoxalase II (GLYII), or glyoxalase III (GLYIII). GLYI and GLYII are the key enzymes of glyoxalase pathways that play an important role in abiotic stress tolerance. Earlier research showed that MG level is lower when both GLYI and GLYII are overexpressed together, compared to GLYI or GLYII single gene overexpressed transgenic plants. D-lactate dehydrogenase (D-LDH) is an integral part of MG detoxification which metabolizes the end product (D-lactate) of the glyoxalase pathway. In this study, two Arabidopsis transgenic lines were constructed using gene pyramiding technique: GLYI and GLYII overexpressed (G-I + II), and GLYI, GLYII, and D-LDH overexpressed (G-I + II + D) plants. G-I + II + D exhibits lower MG and D-lactate levels and enhanced abiotic stress tolerance than the G-I + II and wild-type plants. Further study explores the stress tolerance mechanism of G-I + II + D plants through the interplay of different regulators and plant hormones. This, in turn, modulates the expression of ABA-dependent stress-responsive genes like RAB18, RD22, and RD29B to generate adaptive responses during stress. Therefore, there might be a potential correlation between ABA and MG detoxification pathways. Furthermore, higher STY46, GPX3, and CAMTA1 transcripts were observed in G-I + II + D plants during abiotic stress. Thus, our findings suggest that G-I + II + D has significantly improved MG detoxification, reduced oxidative stress-induced damage, and provided a better protective mechanism against abiotic stresses than G-I + II or wild-type plants. [Display omitted] • Gene pyramiding was done to combine the Glyoxalase pathway along with D-LDH (G-I+II+D). • A comparative analysis was done between G-I+II+D with G-I+II (Glyoxalase I and II overexpressed) transgenics. • Pyramiding D-LDH with glyoxalase pathway (G-I+II+D) enhances abiotic stress tolerance in Arabidopsis thaliana. • G-I+II+D transgenics maintain lower methylglyoxal, D-Lactate, and ROS than G-I+II and wild-type plants during abiotic stress. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MG) is a cytotoxic compound often produced as a side product of metabolic processes such as glycolysis, lipid peroxidation, and photosynthesis. MG is mainly scavenged by the glyoxalase system, a two-step pathway, in which the coordinate activity of GLYI and GLYII transforms it into D-lactate, releasing GSH. In Arabidopsis thaliana, a member of the GLYI family named GLYI4 has been recently characterized. In glyI4 mutant plants, a general stress phenotype characterized by compromised MG scavenging, accumulation of reactive oxygen species (ROS), stomatal closure, and reduced fitness was observed. In order to shed some light on the impact of gly4 loss-of-function on plant metabolism, we applied a high resolution mass spectrometry-based metabolomic approach to Arabidopsis Col-8 wild type and glyI4 mutant plants. A compound library containing a total of 70 metabolites, differentially synthesized in glyI4 compared to Col-8, was obtained. Pathway analysis of the identified compounds showed that the upregulated pathways are mainly involved in redox reactions and cellular energy maintenance, and those downregulated in plant defense and growth. These results improved our understanding of the impacts of glyI4 loss-of-function on the general reprogramming of the plant’s metabolic landscape as a strategy for surviving under adverse physiological conditions.

The dermis is the connective layer between the epidermis and subcutis and harbours nerve endings, glands, blood vessels, and hair follicles. The most abundant cell type is the fibroblast. Dermal fibroblasts have a versatile portfolio of functions within the dermis that correspond with different types of cells by either direct contact or by autocrine and paracrine signalling. Diabetic skin is characterized by itching, numbness, ulcers, eczema, and other pathophysiological changes. These pathogenic phenotypes have been associated with the effects of the reactive glucose metabolite methylglyoxal (MGO) on dermal cells. In this study, dermal fibroblasts were isolated from diabetic and non-diabetic human donors. Cultured dermal fibroblasts from diabetic donors exhibited reduced insulin-induced glucose uptake and reduced expression of the insulin receptor. This diabetic phenotype persists under cell culture conditions. Secretion of IL-6 was increased in fibroblasts from diabetic donors. Increased secretion of IL-6 and MIF was also observed upon the treatment of dermal fibroblasts with MGO, suggesting that MGO is sufficient for triggering these immunomodulatory responses. Remarkably, MIF treatment resulted in decreased activity of MGO-detoxifying glyoxalase-1. Given that reduced glyoxalase activity results in increased MGO levels, these findings suggested a positive-feedback loop for MGO generation, in which MIF, evoked by MGO, in turn blocks MGO-degrading glyoxalase activity. Finally, secretion of procollagen Type I C-Peptide (PICP), a marker of collagen production, was reduced in fibroblast from diabetic donors. Remarkably, treatment of fibroblasts with either MGO or MIF was sufficient for inducing reduced PICP levels. The observations of this study unravel a signalling network in human dermal fibroblasts with the metabolite MGO being sufficient for inflammation and delayed wound healing, hallmarks of T2D.

Aging is a complex natural phenomenon that is manifested by degenerative changes in the structure and function of cells and tissues. D-Galactose-induced aging mice are an artificial accelerated aging model that causes memory and learning impairment, oxidative stress, and neuroinflammation. In this study, we examined the underlying mechanism of an aging mouse model induced by D-galactose. Our behavioral Morris water maze results revealed that D-galactose administration for 2 months significantly induced memory and learning impairment in C57BL/6J mice. High performance liquid chromatography (HPLC) results showed elevated levels of the metabolite methylglyoxal (MG) in D-galactose-induced aging mice. Whether and how D-galactose induces senescence by elevated levels of reactive metabolite MG remain unclear. In our study, MG mainly accumulated through the following two aspects: to increase its source, namely, the triose phosphate produced by the glycolysis pathway, and to reduce its detoxification system, namely, the glyoxalase system. Therefore, elevated MG levels may be one of the causes of brain senescence in D-galactose-induced mice. However, the molecular mechanism of the increased level of the reaction metabolite methylglyoxal requires further exploration.

Aging is a complex natural phenomenon that is manifested by degenerative changes in the structure and function of cells and tissues. D-Galactose-induced aging mice are an artificial accelerated aging model that causes memory and learning impairment, oxidative stress, and neuroinflammation. In this study, we examined the underlying mechanism of an aging mouse model induced by D-galactose. Our behavioral Morris water maze results revealed that D-galactose administration for 2 months significantly induced memory and learning impairment in C57BL/6J mice. High performance liquid chromatography (HPLC) results showed elevated levels of the metabolite methylglyoxal (MG) in D-galactose-induced aging mice. Whether and how D-galactose induces senescence by elevated levels of reactive metabolite MG remain unclear. In our study, MG mainly accumulated through the following two aspects: to increase its source, namely, the triose phosphate produced by the glycolysis pathway, and to reduce its detoxification system, namely, the glyoxalase system. Therefore, elevated MG levels may be one of the causes of brain senescence in D-galactose-induced mice. However, the molecular mechanism of the increased level of the reaction metabolite methylglyoxal requires further exploration. [ABSTRACT FROM AUTHOR]

Detoxification of methylglyoxal, a toxic by‐product of central sugar metabolism, is a major issue for all forms of life. The glyoxalase pathway evolved to effectively convert methylglyoxal into d‐lactate via a glutathione hemithioacetal intermediate. Recently, we have shown that the monomeric glyoxalase I from maize exhibits a symmetric fold with two cavities, potentially harboring two active sites, in analogy with homodimeric enzyme surrogates. Here we confirm that only one of the two cavities exhibits glyoxalase I activity and show that it adopts a tunnel‐shaped structure upon substrate binding. Such conformational change gives rise to independent binding sites for glutathione and methylglyoxal in the same active site, with important implications for the molecular reaction mechanism, which has been a matter of debate for several decades. Database: Structural data are available in The Protein Data Bank database under the accession numbers 6BNN, 6BNX, and 6BNZ. [ABSTRACT FROM AUTHOR]

Key message: FLO15encodes a plastidic glyoxalase I protein, OsGLYI7, which affects compound starch granule formation and starch synthesis in rice endosperm.Abstract: Starch synthesis in rice (Oryza sativa) endosperm is a sophisticated process, and its underlying molecular machinery still remains to be elucidated. Here, we identified and characterized two allelic rice floury endosperm 15 (flo15) mutants, both with a white-core endosperm. The flo15 grains were characterized by defects in compound starch granule development, along with decreased starch content. Map-based cloning of the flo15 mutants identified mutations in OsGLYI7, which encodes a glyoxalase I (GLYI) involved in methylglyoxal (MG) detoxification. The mutations of FLO15/OsGLYI7 resulted in increased MG content in flo15 developing endosperms. FLO15/OsGLYI7 localizes to the plastids, and the in vitro GLYI activity derived from flo15 was significantly decreased relative to the wild type. Moreover, the expression of starch synthesis-related genes was obviously altered in the flo15 mutants. These findings suggest that FLO15 plays an important role in compound starch granule formation and starch synthesis in rice endosperm. [ABSTRACT FROM AUTHOR]

Plant response to salt stress and the mechanism of salt tolerance have received major focus by plant biology researchers. Biotic stresses cause extensive losses in agricultural production globally, but abiotic stress causes significant increase in the methylglyoxal (MG) level of GlyoxalaseI (Gly I). Identification of salt-tolerant genes when characterizing their phenotypes will help to identify novel genes using polymerase chain reaction (PCR) to amplify the DNA coding region for glyoxalase I. This method is specific, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. This method was used rapidly and easily identified glyoxalase I sequences as salt-tolerant genes from Jojoba (Simmondsia chinensis (Link) Schneider). In the present study, the glyoxalase I gene was isolated, amplified by PCR using gene-specific primers and sequenced from the jojoba plant, then compared with other glyoxalase I sequences in other plants and glyoxalase I genes like in Brassica napus, ID: KT720495.1; Brassica juncea ID: Y13239.1, Arachis hypogaea; ID: DQ989209.2; and Arabidopsis thaliana L, ID: AAL84986. The structural gene of glyoxalase I, when sequenced and analyzed, revealed that the uninterrupted open reading frame (ORF) of jojoba Gly I (Jojo-Gly I) spans 775 bp, corresponding to 185 amino acid residues, and shares 45.2% amino acid sequence identity to jojoba (Jojo-Gly I). The cloned ORF, in a multicopy constitutive expression plasmid, complemented the Jojo-Gly I, confirming that the encoded Jojo-Gly I in jojoba showed some homology with other known glyoxalase I sequences of plants.

We have performed quantum mechanics (QM), molecular mechanics (MM) and hybrid QM/MM calculations to study the stereospecific proton exchange of glutathiohydroxyacetone (HOC-SG) by glyoxalase I (GlxI). We did the QM/MM calculations with a large QM system (246 atoms) to investigate the proton-exchange mechanism. Moreover, single-point big-QM energies with 1303 atoms in the big QM system and 22,412 atoms in the MM system were used to compare the energy difference of the stationary structures. GlxI catalyzes the exchange of the pro-S, but not the pro-R hydroxymethyl proton of HOC-SG with a deuterium from the D2O solvent. Classical molecular dynamics simulations with different protonation states of Glu99, Glu172 and HOC-SG led to the determination of most stable species (Glu-172 is protonated and the alcoholic oxygen of HOC-SG is deprotonated). The QM/MM results showed that before binding of HOC-SG, both active-site glutamates are charged, whereas HOC-SG is protonated. When HOC-SG binds, its alcoholic proton (HO) can point toward either Glu-99 or Glu-172. However, if the substrate binds so that HO is directed toward Glu-99, it is not transferred, whereas if it is directed toward Glu-172, the latter abstracts HO. The results showed that transferring HO to the glutamates from the reactant states is the key step to make the proton exchange reaction possible. Our calculations show that order of basicity of the glutamates and HOC-SG inside the enzyme is: Glu-172 > HOC-SG > Glu-99. The calculations allow us to propose a reaction mechanism for the stereospecific proton exchange of HOC-SG by GlxI with an overall barrier of 14.1 kcal/mol.

The Arabidopsis genome annotation include 11 glyoxalase I (GLXI) genes, all encoding for protein members of the vicinal oxygen chelate (VOC) superfamily. The biochemical properties and physiological importance of three Arabidopsis GLXI proteins in the detoxification of reactive carbonyl species has been recently described. Analyses of phylogenetic relationships and conserved GLXI binding sites indicate that the other eight GLXI genes (GLXI-like) do not encode for proteins with GLXI activity. In this perspective article we analyse the structural features of GLXI and GLXI-like proteins, and explore splice forms and transcript abundance under abiotic stress conditions. Finally, we discuss future directions of research on this topic with respect to the substrate identification of GLXI and GLXI-like proteins and the need of reliable quantitative measurements of reactive carbonyl species in plant tissues.

According to an update from the World Health Organization, approximately 50 million people worldwide suffer from epilepsy, and nearly one-third of these individuals are resistant to the currently available antiepileptic drugs, which has resulted in an insistent pursuit of novel strategies for seizure treatment. Recently, methylglyoxal (MG) was demonstrated to serve as a partial agonist of the gamma-aminobutyric acid type A (GABAA) receptor and to play an inhibitory role in epileptic activities. However, MG is also a substrate in the generation of advanced glycation end products (AGEs) that function by activating the receptor of AGEs (RAGE). The AGE/RAGE axis is responsible for the transduction of inflammatory cascades and appears to be an adverse pathway in epilepsy. This study systematically reviewed the significance of GABAergic MG, glyoxalase I (GLO1; responsible for enzymatic catalysis of MG cleavage) and downstream RAGE signaling in epilepsy. This work also discussed the potential of miRNAs that target multiple mRNAs and introduced a preliminary scheme for screening and validating miRNA candidates with the goal of reconciling the conflicting effects of MG for the future development of seizure treatments.

The Arabidopsis genome annotation include 11 glyoxalase I (GLXI) genes, all encoding for protein members of the vicinal oxygen chelate (VOC) superfamily. The biochemical properties and physiological importance of three Arabidopsis GLXI proteins in the detoxification of reactive carbonyl species has been recently described. Analyses of phylogenetic relationships and conserved GLXI binding sites indicate that the other eight GLXI genes (GLXI-like) do not encode for proteins with GLXI activity. In this perspective article we analyse the structural features of GLXI and GLXI-like proteins, and explore splice forms and transcript abundance under abiotic stress conditions. Finally, we discuss future directions of research on this topic with respect to the substrate identification of GLXI and GLXI-like proteins and the need of reliable quantitative measurements of reactive carbonyl species in plant tissues. [ABSTRACT FROM AUTHOR]

Glucose and glucose degradation products (GDPs), contained in peritoneal dialysis (PD) fluids, contribute to the formation of advanced glycation end-products (AGEs). Local damaging effects, resulting in functional impairment of the peritoneal membrane, are well studied. It is also supposed that detoxification of AGE precursors by glyoxalase-1 (GLO1) has beneficial effects on GDP-mediated toxicity. The aim of the current study was to analyze systemic detrimental effects of PD fluids and their prevention by glyoxlase-1. Wild-type and GLO1-overexpressing Caenorhabditis elegans (C. elegans) were cultivated in the presence of low- and high-GDP PD fluids containing 1.5 or 4% glucose. Lifespan, neuronal integrity and neuronal functions were subsequently studied. The higher concentrations of glucose and GDP content resulted in a decrease of maximum lifespan by 2 (P<0.01) and 9 days (P<0.001), respectively. Exposure to low- and high-GDP fluids caused reduction of neuronal integrity by 34 (P<0.05) and 41% (P<0.05). Cultivation of animals in the presence of low-GDP fluid containing 4% glucose caused significant impairment of neuronal function, reducing relative and absolute head motility by 58.5 (P<0.01) and 56.7% (P<0.01), respectively; and relative and absolute tail motility by 55.1 (P<0.05) and 55.0% (P<0.05), respectively. Taken together, GLO1 overexpression protected from glucose-induced lifespan reduction, neurostructural damage and neurofunctional damage under low-GDP-conditions. In conclusion, both glucose and GDP content in PD fluids have systemic impact on the lifespan and neuronal integrity of C. elegans. Detoxification of reactive metabolites by GLO1 overexpression was sufficient to protect lifespan, neuronal integrity and neuronal function in a low-GDP environment. These data emphasize the relevance of the GLO1 detoxifying pathway as a potential therapeutic target in the treatment of reactive metabolite-mediated pathologies. [ABSTRACT FROM AUTHOR]

According to an update from the World Health Organization, approximately 50 million people worldwide suffer from epilepsy, and nearly one-third of these individuals are resistant to the currently available antiepileptic drugs, which has resulted in an insistent pursuit of novel strategies for seizure treatment. Recently, methylglyoxal (MG) was demonstrated to serve as a partial agonist of the gamma-aminobutyric acid type A (GABAA) receptor and to play an inhibitory role in epileptic activities. However, MG is also a substrate in the generation of advanced glycation end products (AGEs) that function by activating the receptor of AGEs (RAGE). The AGE/RAGE axis is responsible for the transduction of inflammatory cascades and appears to be an adverse pathway in epilepsy. This study systematically reviewed the significance of GABAergic MG, glyoxalase I (GLO1; responsible for enzymatic catalysis of MG cleavage) and downstream RAGE signaling in epilepsy. This work also discussed the potential of miRNAs that target multiple mRNAs and introduced a preliminary scheme for screening and validating miRNA candidates with the goal of reconciling the conflicting effects of MG for the future development of seizure treatments. [ABSTRACT FROM AUTHOR]

The effects of Maneb on antioxidant potentials and carbon assimilation were studied in leaves sorghum (Sorghum bicolor L.) plants (C4 species). The plants were sprayed by three doses; recommended dose (2.5 g/L), twice and three times higher at 25, 40 and 55 days after planting (DAP). The leaves were harvested at 5, 10 and 15 days after treatment (DAT) and proteins content, activities of antioxidative enzymes catalase (CAT), guaiacol peroxidase (POD), glutathione reductase (GR), glutathione-S-transferases (GST) and glyoxalase I (GlyI) as well as activity of key enzyme in primary fixation of atmospheric CO2 phosphoenolpyruvate carboxylase (PEPC) and chlorophyll content were estimated. The protein content was found increased under Maneb treatment. While the POD and GR activities appear unaffected, CAT activity increased at 10 and 15 DAT. Whereas the GST activity increased significantly in plants treated with 5 g/L at 5 DAT and in plant exposed to 2.5 g/L at 10 DAT. The activity and proteins levels of GlyI were increased progressively with increasing fungicide treatment. This increase was more pronounced at 5 DAT; being 1.7 and 1.5 fold for 5 and 7.5 g/L, respectively. However, both PEPC activity and chlorophyll content seem unchanged in presence of fungicide even at high concentration. These results indicate that sorghum leaves have a well-developed systems of protection from carbonyl stress. [ABSTRACT FROM AUTHOR]

Many cancer cells undergo metabolic reprogramming known as the Warburg effect, which is characterized by a greater dependence on glycolysis for ATP generation, even under normoxic conditions. Glyoxalase I (GLO I) is a rate-limiting enzyme involved in the detoxification of cytotoxic methylglyoxal formed in glycolysis and which is known to be highly expressed in many cancer cells. Thus, specific inhibitors of GLO I are expected to be effective anticancer drugs. We previously discovered a novel GLO I inhibitor named TLSC702. Although the strong inhibitory activity of TLSC702 was observed in the in vitro enzyme assay, higher concentrations were required to induce apoptosis at the cellular level. One of the proposed reasons for this difference is that cancer cells alter the energy metabolism leading them to become more dependent on mitochondrial respiration than glycolysis ( Metabolic shift ) to avoid apoptosis induction. Thus, we assumed that combination of TLSC702 with shikonin—a specific inhibitor of pyruvate kinase M2 (PKM2) that acts as a driver of TCA cycle by supplying pyruvate and which is known to be specifically expressed in cancer cells—would have anticancer effects. We herein show the anticancer effects of combination treatment with TLSC702 and shikonin, and a possible anticancer mechanism. [ABSTRACT FROM AUTHOR]

Objectives: To study expression of glyoxalase I in patients of diabetic retinopathy. Methods: This cross-sectional comparative study was conducted at Centre for Research in Experimental and Applied Medicine (CREAM), Department of Biochemistry and Molecular Biology, Army Medical College, Rawalpindi in collaboration with Armed Forces Institute of Ophthalmology (AFIO) from January 2015 to November 2015. Sampling technique was non- probability purposive sampling. Total 60 subjects were enrolled in two groups. Group-I comprised 30 patients of diabetic retinopathy and Group-II of 30 normal healthy controls. Clinical and demographic data was collected and fasting venous blood samples (2 ml) were drawn. RNA was extracted and subjected to cDNA synthesis. Expression analysis for glyoxalase I was carried out and relative quantification done by double delta Ct method. Results: Mean age of the patients was 61.30 ± 7.06 years and mean age of controls was 59.60 ± 6.43 years. There were 17 (56.7%) males and 13 (43.3%) females in Group-I while Group-II comprised 14 (46.7%) males and 16 (53.3%) females. There was down regulation of glyoxalase I among patients of diabetic retinopathy in comparison with controls when relative gene expression was calculated. Conclusion: Down regulation of glyoxalase I in patients of diabetic retinopathy suggests it to be a contributory factor in the development of disease. [ABSTRACT FROM AUTHOR]

Erypoegin K is an isoflavone isolated from the stem bark of Erythrina poeppigiana. It contains a furan group at the A-ring of the core isoflavone structure and can inhibit the activity of glyoxalase I, an enzyme that catalyzes the detoxification of methylglyoxal (MG), a by-product of glycolysis. In the present study, we found that erypoegin K has a potent cytotoxic effect on human leukemia HL-60 cells. Its cytotoxic effect was much stronger than that of a known glyoxalase I inhibitor S- p-bromobenzylglutathione cyclopentyl diester. Conversely, erypoegin K demonstrated weak cytotoxicity toward normal human peripheral lymphocytes. The treatment of HL-60 cells with erypoegin K significantly induced caspase-3 activity, whereas the pretreatment of the cells with caspase-3 inhibitor suppressed erypoegin K-induced cell death. Furthermore, nuclear condensation and apoptotic genome DNA fragmentation were observed in erypoegin K-treated HL-60 cells. These results indicated that the observed cell death was mediated by apoptosis. In addition, the toxic compound MG was highly accumulated in the culture medium of erypoegin K-treated HL-60 cells, suggesting that cell apoptosis was triggered by extracellular MG. The present study showed that erypoegin K has a potent apoptosis-inducing effect on cancerous cell lines, such as HL-60. [ABSTRACT FROM AUTHOR]

Objectives: Non-enzymatic glycation is closely associated with altered mechanical properties of connective tissue. Pregnancy, marked with high levels of female hormones, confers unique alteration to the mechanical properties of pelvic connective tissues in order to meet their physiological demands. However, there are few studies on glycation content and its influence on the mechanical properties of pelvic connective tissues during pregnancy. We hypothesise that the glycation content in pelvic tissues will change with a corresponding alteration in their mechanical properties, and that these changes are influenced by hormone levels. This study aims to investigate the correlation of vaginal tissue glycation content and mechanical property changes during pregnancy in association with the expression of a key pregnancy hormone (oestrogen) receptor, and an antioxidant enzyme, glyoxalase I.Study Design: A rat vaginal tissue model (tissues from non-pregnant and E15-E18 (last trimester) pregnant rats) was used in this study. Mechanical characteristics of vaginal tissues were analysed by a ball-indentation technique while modulus and morphology of the collagen fibrils within the tissues were measured with atomic force microscopy. A glycation marker, pentosidine, was quantified by a high performance liquid chromatography. The expression of oestrogen receptor and glyoxalase I in the tissue was qualified by immunochemical staining. The glycosaminoglycan (GAG) concentration difference in the tissues were quantified by a biochemical assay.Results: Pregnant rat vaginal tissue was characterised by significantly lower amounts of pentosidine, higher oestrogen receptor and glyoxalase I expression with larger creep, lower elastic modulus, larger fibril diameter and higher GAG content than their non-pregnant counterpart. There was a negative correlation between pentosidine and vaginal tissue creep.Conclusion: There was a reduction in vaginal tissue pentosidine in pregnancy with an associated increase in oestrogen receptor and glyoxalase I immunoexpression. Reduced glycation was associated with increased creeping of vaginal tissue. Oestrogen may therefore play a role in the increase of the vaginal wall's capacity to stretch through glyoxalase I up-regulation and subsequent glycation reduction. The new insight of the correlation of women's oestrogen level, glycation reaction and pelvic tissue mechanical property from this study may enhance our understanding of some pelvic organ diseases. [ABSTRACT FROM AUTHOR]

Human glyoxalase I (GLO I), a rate-limiting enzyme for detoxification of methylglyoxal (MG), a by-product of glycolysis, is known to be a potential therapeutic target for cancer. Here, we searched new scaffolds from natural compounds for designing novel GLO I inhibitors and found trans -stilbene scaffold. We examined the inhibitory abilities to human GLO I of commercially available trans- stilbene compounds. Among them, piceatannol was found to have the most potent inhibitory activity against human GLO I. Piceatannol could inhibit the proliferation of human lung cancer NCI-H522 cells, which are dependent on GLO I for survival, in a dose- and time-dependent manner. In addition, piceatannol more significantly inhibited the proliferation of NCI-H522 cells than that of NCI-H460 cells, which are less dependent on GLO I. Importantly, overexpression of GLO I in NCI-H522 cells resulted in less sensitive to the antiproliferative activity of piceatannol. Taken together, this is the first report demonstrating that piceatannol inhibits GLO I activity and the GLO I-dependent proliferation of cancer cells. Furthermore, we determined a pharmacophore for novel inhibitors of human GLO I by computational simulation analyses of the binding mode of piceatannol to the enzyme hot spot in the active site. We suggest that piceatannol is a possible lead compound for the development of novel GLO I inhibitory anticancer drugs. [ABSTRACT FROM AUTHOR]

The cellular levels of methylglyoxal (MG), a toxic byproduct of glycolysis, rise under various abiotic stresses in plants. Detoxification of MG is primarily through the glyoxalase pathway. The first enzyme of the pathway, glyoxalase I (GLYI), is a cytosolic metalloenzyme requiring either Ni2+ or Zn2+ for its activity. Plants possess multiple GLYI genes, of which only some have been partially characterized; hence, the precise molecular mechanism, subcellular localization and physiological relevance of these diverse isoforms remain enigmatic. Here, we report the biochemical properties and physiological role of a putative chloroplast-localized GLYI enzyme, OsGLYI-8, from rice, which is strikingly different from all hitherto studied GLYI enzymes in terms of its intracellular localization, metal dependency and kinetics. In contrast to its predicted localization, OsGLYI-8 was found to localize in the nucleus along with its substrate, MG. Further, OsGLYI-8 does not show a strict requirement for metal ions for its activity, is functional as a dimer and exhibits unusual biphasic steady-state kinetics with a low-affinity and a high-affinity substrate-binding component. Loss of AtGLYI-2, the closest Arabidopsis ortholog of OsGLYI-8, results in severe germination defects in the presence of MG and growth retardation under salinity stress conditions. These defects were rescued upon complementation with AtGLYI-2 or OsGLYI-8. Our findings thus provide evidence for the presence of a GLYI enzyme and MG detoxification in the nucleus. [ABSTRACT FROM AUTHOR]

Glyoxalase I (Gly I) is a component of the glyoxalase system which is involved in the detoxification of methylglyoxal, a byproduct of glycolysis. In the present study, a gene of rice ( Oryza sativa L., cv. Nipponbare) encoding Gly I was cloned and characterized. The quantitative real-time PCR analysis indicated that rice Gly I ( OsGly I ) was ubiquitously expressed in root, stem, leaf, leaf sheath and spikelet with varying abundance. OsGly I was markedly upregulated in response to NaCl, ZnCl 2 and mannitol in rice seedlings. For further functional investigation, OsGly I was overexpressed in rice using Agrobacterium- mediated transformation. Transgenic rice lines exhibited increased glyoxalase enzyme activity, decreased methylglyoxal level and improved tolerance to NaCl, ZnCl 2 and mannitol compared to wild-type plants. Enhancement of stress tolerance in transgenic lines was associated with reduction of malondialdehyde content which was derived from cellular lipid peroxidation. In addition, the OsGly I -overexpression transgenic plants performed higher seed setting rate and yield. Collectively, these results indicate the potential of bioengineering the Gly I gene in crops. [ABSTRACT FROM AUTHOR]

Density functional theory has been used to study the mechanism and stereospecificity of the catalytic reaction of human glyoxalase I. We used the quantum mechanical cluster method to model the enzyme active site. Glyoxalase I accepts both enantiomers of the hemithioacetal between methylglyoxal and glutathione and converts them to the S -D enantiomer of lactoylglutathione. We have compared several previously suggested or alternative reaction mechanisms for both substrates on an equal footing. The results show that the coordination shell of the Zn ion in the optimized geometries is more symmetric than in some inhibitor crystal structures, which we assign to differences in the electronic structure and the protonation states of the substrate. The symmetry of the active site model indicates that the enzyme can use the same reaction mechanism for the S and the R enantiomers of the substrate, but with exchanged roles of the two active-site glutamate residues. However, the calculations show some asymmetry (0–4 kcal mol −1 differences in reaction energies and activation barriers), caused by the different coordination states of the glutamate residues in the starting crystal structure. Our results indicate that the only possibility for the stereospecificity of glyoxalase I is differences in the electrostatic surroundings and flexibility of the glutamate residues in the active site owing to their neighboring residues in the protein. [ABSTRACT FROM AUTHOR]