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The EMBO Journal Jan 2018Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity...
Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.
Topics: Animals; COS Cells; Chlorocebus aethiops; Drosophila melanogaster; Fixatives; Formaldehyde; Glyoxal; HeLa Cells; Humans; Immunohistochemistry; Mice; Microscopy, Fluorescence; Nerve Tissue Proteins; Tissue Fixation
PubMed: 29146773
DOI: 10.15252/embj.201695709 -
Cell Chemical Biology Feb 2020Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular...
Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys" modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions.
Topics: Alkynes; Glutathione; Glycolysis; Glycosylation; HEK293 Cells; Humans; Lactoylglutathione Lyase; Lysine; Pyruvaldehyde; Recombinant Proteins; Thiolester Hydrolases
PubMed: 31767537
DOI: 10.1016/j.chembiol.2019.11.005 -
The Plant Cell Apr 2023High salinity, an adverse environmental factor affecting about 20% of irrigated arable land worldwide, inhibits plant growth and development by causing oxidative stress,...
High salinity, an adverse environmental factor affecting about 20% of irrigated arable land worldwide, inhibits plant growth and development by causing oxidative stress, damaging cellular components, and disturbing global metabolism. However, whether and how reactive oxygen species disturb the metabolism of salt-stressed plants remain elusive. Here, we report that salt-induced hydrogen peroxide (H2O2) inhibits the activity of plastid triose phosphate isomerase (pdTPI) to promote methylglyoxal (MG) accumulation and stimulates the sulfenylation of pdTPI at cysteine 74. We also show that MG is a key factor limiting the plant growth, as a decrease in MG levels completely rescued the stunted growth and repressed salt stress tolerance of the pdtpi mutant. Furthermore, targeting CATALASE 2 into chloroplasts to prevent salt-induced overaccumulation of H2O2 conferred salt stress tolerance, revealing a role for chloroplastic H2O2 in salt-caused plant damage. In addition, we demonstrate that the H2O2-mediated accumulation of MG in turn induces H2O2 production, thus forming a regulatory loop that further inhibits the pdTPI activity in salt-stressed plants. Our findings, therefore, illustrate how salt stress induces MG production to inhibit the plant growth.
Topics: Hydrogen Peroxide; Pyruvaldehyde; Salt Stress; Oxidative Stress; Plants; Chloroplasts; Stress, Physiological
PubMed: 36695476
DOI: 10.1093/plcell/koad019 -
Drug Metabolism and Drug Interactions 2008Glycation of proteins, nucleotides and basic phospholipids by glyoxal and methylglyoxal--physiological substrates of glyoxalase 1--is potentially damaging to the... (Review)
Review
Glycation of proteins, nucleotides and basic phospholipids by glyoxal and methylglyoxal--physiological substrates of glyoxalase 1--is potentially damaging to the proteome, genome and lipidome. Glyoxalase 1 suppresses glycation by these alpha-oxoaldehyde metabolites and thereby represents part of the enzymatic defence against glycation. Albert Szent-Györgyi pioneered and struggled to understand the physiological function of methylglyoxal and the glyoxalase system. We now appreciate that glyoxalase 1 protects against dicarbonyl modifications of the proteome, genome and lipome. Latest research suggests there are functional modifications of this process--implying a role in cell signalling, ageing and disease.
Topics: Aging; Animals; Drug Resistance, Neoplasm; Glycation End Products, Advanced; Glyoxal; Humans; Lactoylglutathione Lyase; Nucleotides; Proteins; Pyruvaldehyde; Thiolester Hydrolases
PubMed: 18533367
DOI: 10.1515/dmdi.2008.23.1-2.125 -
International Journal of Molecular... Jan 2017Glyoxal (GO) and methylglyoxal (MG), belonging to α-oxoaldehydes, are produced by organisms from bacteria to humans by glucose oxidation, lipid peroxidation, and DNA... (Review)
Review
Glyoxal (GO) and methylglyoxal (MG), belonging to α-oxoaldehydes, are produced by organisms from bacteria to humans by glucose oxidation, lipid peroxidation, and DNA oxidation. Since glyoxals contain two adjacent reactive carbonyl groups, they are referred to as reactive electrophilic species (RES), and are damaging to proteins and nucleotides. Therefore, glyoxals cause various diseases in humans, such as diabetes and neurodegenerative diseases, from which all living organisms need to be protected. Although the glyoxalase system has been known for some time, details on how glyoxals are sensed and detoxified in the cell have not been fully elucidated, and are only beginning to be uncovered. In this review, we will summarize the current knowledge on bacterial responses to glyoxal, and specifically focus on the glyoxal-associated regulators YqhC and NemR, as well as their detoxification mediated by glutathione (GSH)-dependent/independent glyoxalases and NAD(P)H-dependent reductases. Furthermore, we will address questions and future directions.
Topics: Aldehyde Oxidoreductases; Bacteria; Bacterial Proteins; Glutathione; Glyoxal; Inactivation, Metabolic; Lactoylglutathione Lyase; Oxidative Stress; Pyruvaldehyde; Thiolester Hydrolases
PubMed: 28106725
DOI: 10.3390/ijms18010169 -
Biomolecular Concepts Dec 2015The glyoxalase enzyme system utilizes intracellular thiols such as glutathione to convert α-ketoaldehydes, such as methylglyoxal, into D-hydroxyacids. This overview... (Review)
Review
The glyoxalase enzyme system utilizes intracellular thiols such as glutathione to convert α-ketoaldehydes, such as methylglyoxal, into D-hydroxyacids. This overview discusses several main aspects of the glyoxalase system and its likely function in the cell. The control of methylglyoxal levels in the cell is an important biochemical imperative and high levels have been associated with major medical symptoms that relate to this metabolite's capability to covalently modify proteins, lipids and nucleic acid.
Topics: Catalytic Domain; Crystallography, X-Ray; Glutathione; Humans; Kinetics; Lactoylglutathione Lyase; Models, Molecular; Molecular Structure; Pyruvaldehyde; Thiolester Hydrolases
PubMed: 26552067
DOI: 10.1515/bmc-2015-0025 -
International Journal of Molecular... Mar 2022Glyoxal (GO) and methylglyoxal (MGO) are highly reactive species formed in carbohydrate metabolism. -Carboxymethyllysine (CML) and -carboxyethyllysine (CEL) are...
Free L-Lysine and Its Methyl Ester React with Glyoxal and Methylglyoxal in Phosphate Buffer (100 mM, pH 7.4) to Form -Carboxymethyl-Lysine, -Carboxyethyl-Lysine and -Hydroxymethyl-Lysine.
Glyoxal (GO) and methylglyoxal (MGO) are highly reactive species formed in carbohydrate metabolism. -Carboxymethyllysine (CML) and -carboxyethyllysine (CEL) are considered to be the advanced glycation end-products (AGEs) of L-lysine (Lys) with GO and MGO, respectively. Here, we investigated the reaction of free L-lysine (Lys) with GO and MGO in phosphate buffer (pH 7.4) at 37 °C and 80 °C in detail in the absence of any other chemicals which are widely used to reduce Schiff bases. The concentrations of Lys, GO and MGO used in the experiments were 0.5, 2.5, 5.0, 7.5 and 10 mM. The reaction time ranged between 0 and 240 min. Experiments were performed in triplicate. The concentrations of remaining Lys and of CML and CEL formed in the reaction mixtures were measured by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS). Our experiments showed that CML and CEL were formed at higher concentrations at 80 °C compared to 37 °C. CML was found to be the major reaction product. In mixtures of GO and MGO, MGO inhibited the formation of CML from Lys (5 mM) in a concentration-dependent manner. The highest CML concentration was about 300 µM corresponding to a reaction yield of 6% with respect to Lys. An addition of Lys to GO, MGO and their mixtures resulted in strong reversible decreases in the Lys concentration up to 50%. It is assumed that free Lys reacts rapidly with GO and MGO to form many not yet identified reaction products. Reaction mixtures of Lys and MGO were stronger colored than those of Lys and GO, notably at 80 °C, indicating higher reactivity of MGO towards Lys that leads to polymeric colored MGO species. We have a strong indication of the formation of -(hydroxymethyl)-lysine (HML) as a novel reaction product of Lys methyl ester with MGO. A mechanism is proposed for the formation of HML from Lys and MGO. This mechanism may explain why Lys and GO do not react to form a related product. Preliminary analyses show that HML is formed at higher concentrations than CEL from Lys methyl ester and MGO. No Schiff bases or their hydroxylic precursors were identified as reaction products. In their reactions with Lys, GO and MGO are likely to act both as chemical oxidants on the terminal aldehyde group to a carboxylic group (i.e., R-CHO to R-COOH) and as chemical reductors on labile Schiff bases (R-CH=N-R to R-CH-NH-R) presumably via disproportionation and hydride transfer. Our study shows that free non-proteinic Lys reacts with GO and MGO to form CML, CEL and HML in very low yield. Whether proteinic Lys also reacts with MGO to form HML residues in proteins remains to be investigated. The physiological occurrence and concentration of HML in biological fluids and tissues and its relation to CML and CEL are elusive and warrant further investigations in health and disease. Chemical synthesis and structural characterization of HML are expected to advance and accelerate the scientific research in this topic.
Topics: Esters; Gas Chromatography-Mass Spectrometry; Glycation End Products, Advanced; Glyoxal; Hydrogen-Ion Concentration; Lysine; Magnesium Oxide; Phosphates; Pyruvaldehyde; Schiff Bases
PubMed: 35408807
DOI: 10.3390/ijms23073446 -
Chemical Research in Toxicology Oct 2022Metabolism is an essential part of life that provides energy for cell growth. During metabolic flux, reactive electrophiles are produced that covalently modify... (Review)
Review
Metabolism is an essential part of life that provides energy for cell growth. During metabolic flux, reactive electrophiles are produced that covalently modify macromolecules, leading to detrimental cellular effects. Methylglyoxal (MG) is an abundant electrophile formed from lipid, protein, and glucose metabolism at intracellular levels of 1-4 μM. MG covalently modifies DNA, RNA, and protein, forming advanced glycation end products (MG-AGEs). MG and MG-AGEs are associated with the onset and progression of many pathologies including diabetes, cancer, and liver and kidney disease. Regulating MG and MG-AGEs is a potential strategy to prevent disease, and they may also have utility as biomarkers to predict disease risk, onset, and progression. Here, we review recent advances and knowledge surrounding MG, including its production and elimination, mechanisms of MG-AGEs formation, the physiological impact of MG and MG-AGEs in disease onset and progression, and the latter in the context of its receptor RAGE. We also discuss methods for measuring MG and MG-AGEs and their clinical application as prognostic biomarkers to allow for early detection and intervention prior to disease onset. Finally, we consider relevant clinical applications and current therapeutic strategies aimed at targeting MG, MG-AGEs, and RAGE to ultimately improve patient outcomes.
Topics: Glucose; Glycation End Products, Advanced; Humans; Lipids; Pyruvaldehyde; RNA; Receptor for Advanced Glycation End Products
PubMed: 36197742
DOI: 10.1021/acs.chemrestox.2c00160 -
PloS One 2017Tissue fixation in phosphate buffered formalin (PBF) remains the standard procedure in histopathology, since it results in an optimal structural, antigenic and molecular...
Tissue fixation in phosphate buffered formalin (PBF) remains the standard procedure in histopathology, since it results in an optimal structural, antigenic and molecular preservation that justifies the pivotal role presently played by diagnoses on PBF-fixed tissues in precision medicine. However, toxicity of formaldehyde causes an environmental concern and may demand substitution of this reagent. Having observed that the reported drawbacks of commercially available glyoxal substitutes of PBF (Prefer, Glyo-fix, Histo-Fix, Histo-CHOICE, and Safe-Fix II) are likely related to their acidity, we have devised a neutral fixative, obtained by removing acids from the dialdehyde glyoxal with an ion-exchange resin. The resulting glyoxal acid-free (GAF) fixative has been tested in a cohort of 30 specimens including colon (N = 25) and stomach (N = 5) cancers. Our results show that GAF fixation produces a tissue and cellular preservation similar to that produced by PBF. Comparable immuno-histochemical and molecular (DNA and RNA) analytical data were obtained. We observed a significant enrichment of longer DNA fragment size in GAF-fixed compared to PBF-fixed samples. Adoption of GAF as a non-toxic histological fixative of choice would require a process of validation, but the present data suggest that it represents a reliable candidate.
Topics: DNA; Fixatives; Formaldehyde; Glyoxal; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; RNA; Sequence Analysis, DNA; Tissue Fixation
PubMed: 28796828
DOI: 10.1371/journal.pone.0182965 -
Molecules (Basel, Switzerland) Nov 2022Advances in molecular biology technology have piqued tremendous interest in glycometabolism and bioenergetics in homeostasis and neural development linked to ageing and... (Review)
Review
Advances in molecular biology technology have piqued tremendous interest in glycometabolism and bioenergetics in homeostasis and neural development linked to ageing and age-related diseases. Methylglyoxal (MGO) is a by-product of glycolysis, and it can covalently modify proteins, nucleic acids, and lipids, leading to cell growth inhibition and, eventually, cell death. MGO can alter intracellular calcium homeostasis, which is a major cell-permeant precursor to advanced glycation end-products (AGEs). As side-products or signalling molecules, MGO is involved in several pathologies, including neurodevelopmental disorders, ageing, and neurodegenerative diseases. In this review, we demonstrate that MGO (the metabolic side-product of glycolysis), the GLO system, and their analogous relationship with behavioural phenotypes, epigenetics, ageing, pain, and CNS degeneration. Furthermore, we summarise several therapeutic approaches that target MGO and the glyoxalase (GLO) system in neurodegenerative diseases.
Topics: Pyruvaldehyde; Lactoylglutathione Lyase; Magnesium Oxide; Glycolysis; Brain
PubMed: 36432007
DOI: 10.3390/molecules27227905