Phenotypic and genome-wide studies on dicarbonyls: major associations to glomerular filtration rate and gamma-glutamyltransferase activity.

BACKGROUND: The dicarbonyl compounds methylglyoxal (MG), glyoxal (GO) and 3-deoxyglucosone (3-DG) have been linked to various diseases. However, disease-independent phenotypic and genotypic association studies with phenome-wide and genome-wide reach, respectively, have not been provided. METHODS: MG, GO and 3-DG were measured by LC-MS in 1304 serum samples of two populations (KORA, n = 482; BiDirect, n = 822) and assessed for associations with genome-wide SNPs (GWAS) and with phenome-wide traits. Redundancy analysis (RDA) was used to identify major independent trait associations. FINDINGS: Mutual correlations of dicarbonyls were highly significant, being stronger between MG and GO (ρ = 0.6) than between 3-DG and MG or GO (ρ = 0.4). Significant phenotypic results included associations of all dicarbonyls with sex, waist-to-hip ratio, glomerular filtration rate (GFR), gamma-glutamyltransferase (GGT), and hypertension, of MG and GO with age and C-reactive protein, of GO and 3-DG with glucose and antidiabetics, of MG with contraceptives, of GO with ferritin, and of 3-DG with smoking. RDA revealed GFR, GGT and, in case of 3-DG, glucose as major contributors to dicarbonyl variance. GWAS did not identify genome-wide significant loci. SNPs previously associated with glyoxalase activity did not reach nominal significance. When multiple testing was restricted to the lead SNPs of GWASs on the traits selected by RDA, 3-DG was found to be associated (p = 2.3 × 10-5) with rs1741177, an eQTL of NF-κB inhibitor NFKBIA. INTERPRETATION: This large-scale, population-based study has identified numerous associations, with GFR and GGT being of pivotal importance, providing unbiased perspectives on dicarbonyls beyond the current state. FUNDING: Deutsche Forschungsgemeinschaft, Helmholtz Munich, German Centre for Cardiovascular Research (DZHK), German Federal Ministry of Research and Education (BMBF).

Structure-guided approach to modify the substrate specificity of the protein human deglycase-1 (hDJ-1).

Glycation is a non-enzymatic reaction wherein sugars or dicarbonyls such as methylglyoxal (MGO) and glyoxal (GO) react with proteins, leading to protein inactivation. The hydrolysing enzyme human deglycase-1 (hDJ-1) is reported to decrease glycative stress by deglycating the modified proteins, specifically at cysteine, lysine, and arginine sites. This specificity of hDJ-1 is thought to be regulated by its active site cysteine residue (Cys106). Structural analysis of hDJ-1 by molecular docking and simulation studies, however, indicates a possible role of glutamate (Glu18) in determining its substrate specificity. To elucidate this, Glu18 present at the catalytic site of hDJ-1 was modified to aspartate (Asp18) by SDM, and the resultant mutant was termed mutant DJ-1 (mDJ-1). Both hDJ-1 and mDJ-1 were heterologously expressed in Escherichia coli BL21 (DE3) strain and purified to homogeneity. The hDJ-1 showed kcat values of 1.45 × 103 s-1, 3.6 × 102 s-1, and 3.1 × 102 s-1, and Km values 0.181 mM, 18.18 mM, and 12.5 mM for N-acetylcysteine (NacCys), N-acetyllysine (NacLys), and N-acetylarginine (NacArg), respectively. The mDJ-1 showed altered kcat values (8 × 102 s-1, 3.8 × 102 s-1, 4.9 × 102 s-1) and Km values of 0.14 mM, 6.25 mM, 5.88 mM for NacCys, NacLys and NacArg, respectively. A single amino acid change (Glu18 to Asp18) improved the substrate specificity of mDJ-1 toward NacLys and NacArg. Understanding hDJ-1's structure and enhanced functionality will facilitate further exploration of its therapeutic potential for the treatment of glycation-induced diabetic complications.

Induction of vascular endothelial growth factor-A165a in human retinal and endothelial cells in response to glyoxal.

Low-density lipoprotein (LDL) apheresis is effective and safe for patients with diabetes, proteinuria, and dyslipidemia. Diabetes mellitus is accompanied by ocular microvascular complications like retinal neovascularization or diabetic macular edema. These are leading causes of blindness and can be mediated by abnormal vessel growth and increased vascular permeability due to elevated levels of vascular endothelial growth factor (VEGF) in diabetic patients. In this study, we established methods to study the expression of different VEGF isoforms in human retinal and endothelial cells. The VEGF-A165a isoform is much higher expressed in retinal cells, compared to endothelial cells. Stimulation with glyoxal as a model of oxidative stress under diabetic conditions lead to a pronounced induction of VEGF-A165a in human retinal and endothelial cells. These data suggest that diabetes and oxidative stress induce VEGF-A isoforms which could be relevant in regulating the ingrowths of novel blood vessels into the retina in diabetic patients.

Investigating the molecular mechanisms of glyoxal-induced cytotoxicity in human embryonic kidney cells: Insights from network toxicology and cell biology experiments.

Glyoxal, a reactive carbonyl species, can be generated both endogenously (glucose metabolism) and exogenously (cigarette smoke and food system). Increasing evidence demonstrates that glyoxal exacerbates the development and progression of diabetic nephropathy, but the underlying mechanisms of glyoxal toxicity to human embryonic kidney (HEK293) cells remain unclear. In this work, the molecular mechanisms of glyoxal-induced cytotoxicity in HEK293 cells were explored with network toxicology and cell biology experiments. Network toxicology results showed that oxidative stress and advanced glycation end products (AGEs)/RAGE signaling pathways played a crucial role in glyoxal toxicity. Next, further validation was performed at the cellular level. Glyoxal activated the AGEs-RAGE signaling pathway, caused the increase of cellular ROS, and activated the p38MAPK and JNK signaling pathways, causing cellular oxidative stress. Furthermore, glyoxal caused the activation of the NF-κB signaling pathway and increased the expression of TGF-ß1, indicating that glyoxal caused cellular inflammation. Moreover, glyoxal caused cellular DNA damage accompanied by the activation of DNA damage response pathways. Finally, the mitochondrial apoptosis pathway was activated. The results that obtained in cell biology were consistent with network toxicology, which corroborated each other and together indicated that glyoxal induced HEK293 cells damage via the process of oxidative stress, the AGEs-RAGE pathway, and their associated signaling pathways. This study provides the experimental basis for the cytotoxicity of glyoxal on HEK293 cells.

The possible role of methylglyoxal metabolism in cancer.

Tumours reprogram their metabolism to acquire an evolutionary advantage over normal cells. However, not all such metabolic pathways support energy production. An example of these metabolic pathways is the Methylglyoxal (MG) one. This pathway helps maintain the redox state, and it might act as a phosphate sensor that monitors the intracellular phosphate levels. In this work, we discuss the biochemical step of the MG pathway and interrelate it with cancer.

The Role of Glyoxalase in Glycation and Carbonyl Stress Induced Metabolic Disorders.

Glycation refers to the covalent binding of sugar molecules to macromolecules, such as DNA, proteins, and lipids in a non-enzymatic reaction, resulting in the formation of irreversibly bound products known as advanced glycation end products (AGEs). AGEs are synthesized in high amounts both in pathological conditions, such as diabetes and under physiological conditions resulting in aging. The body's anti-glycation defense mechanisms play a critical role in removing glycated products. However, if this defense system fails, AGEs start accumulating, which results in pathological conditions. Studies have been shown that increased accumulation of AGEs acts as key mediators in multiple diseases, such as diabetes, obesity, arthritis, cancer, atherosclerosis, decreased skin elasticity, male erectile dysfunction, pulmonary fibrosis, aging, and Alzheimer's disease. Furthermore, glycation of nucleotides, proteins, and phospholipids by α-oxoaldehyde metabolites, such as glyoxal (GO) and methylglyoxal (MGO), causes potential damage to the genome, proteome, and lipidome. Glyoxalase-1 (GLO-1) acts as a part of the anti-glycation defense system by carrying out detoxification of GO and MGO. It has been demonstrated that GLO-1 protects dicarbonyl modifications of the proteome and lipidome, thereby impeding the cell signaling and affecting age-related diseases. Its relationship with detoxification and anti-glycation defense is well established. Glycation of proteins by MGO and GO results in protein misfolding, thereby affecting their structure and function. These findings provide evidence for the rationale that the functional modulation of the GLO pathway could be used as a potential therapeutic target. In the present review, we summarized the newly emerged literature on the GLO pathway, including enzymes regulating the process. In addition, we described small bioactive molecules with the potential to modulate the GLO pathway, thereby providing a basis for the development of new treatment strategies against age-related complications.

Glycation and Antioxidants: Hand in the Glove of Antiglycation and Natural Antioxidants.

The non-enzymatic interaction of sugar and protein resulting in the formation of advanced glycation end products responsible for cell signaling alterations ultimately leads to the human chronic disorders such as diabetes mellitus, cardiovascular diseases, cancer, etc. Studies suggest that AGEs upon interaction with receptors for advanced glycation end products (RAGE) result in the production of pro-inflammatory molecules and free radicals that exert altered gene expression effect. To date, many studies unveiled the potent role of synthetic and natural agents in inhibiting the glycation reaction at a lesser or greater extent. This review focuses on the hazards of glycation reaction and its inhibition by natural antioxidants, including polyphenols.

Dicarbonyls Generation, Toxicities, Detoxifications and Potential Roles in Diabetes Complications.

It has been well established that advanced glycation end-products (AGEs) have a strong correlation with diabetes and its secondary complications. Moreover, dicarbonyls, especially, methylglyoxal (MG) and glyoxal, accelerate AGEs formation and hence, have potential roles in the pathogenesis of diabetes. They can also induce oxidative stress and concomitantly decrease the efficiency of antioxidant enzymes. Increased proinflammatory cytokines (tumor necrosis factor-α and interleukin- 1ß) are secreted by monocytes due to the dicarbonyl-modified proteins. High levels of blood dicarbonyls have been identified in diabetes and its associated complications (retinopathy, nephropathy and neuropathy). This review aims to provide a better understanding by including in-depth information about the formation of MG and glyoxal through multiple pathways with a focus on their biological functions and detoxifications. The potential role of these dicarbonyls in secondary diabetic complications is also discussed.

Glyoxalase System in the Progression of Skin Aging and Skin Malignancies.

Dicarbonyl compounds, including methylglyoxal (MGO) and glyoxal (GO), are mainly formed as byproducts of glucose metabolism. The main glyoxalase system consists of glyoxalase I and II (Glo1 and Glo2) and is the main enzyme involved in the detoxification of dicarbonyl stress, which occurs as an accumulation of MGO or GO due to decreased activity or expression of Glo1. Dicarbonyl stress is a major cause of cellular and tissue dysfunction that causes various health issues, including diabetes, aging, and cancer. The skin is the largest organ in the body. In this review, we discuss the role of the glyoxalase system in the progression of skin aging, and more importantly, skin malignancies. We also discuss the future prospects of the glyoxalase system in other skin abnormalities such as psoriasis and vitiligo, including hyperpigmentation. Finally, in the present review, we suggest the role of glyoxalase in the progression of skin aging and glyoxalase system as a potential target for anticancer drug development for skin cancer.

Degradation of Peptide-Bound Maillard Reaction Products in Gastrointestinal Digests of Glyoxal-Glycated Casein by Human Colonic Microbiota.

A large portion of Maillard reaction products (MRPs) cannot be absorbed in the upper gut and therefore may be further decomposed and utilized by colonic microbiota (CM). This work reported the stability of UV-absorbent MRPs, fluorescent MRPs and peptide-bound N(ε)-(carboxymethyl)-lysine (CML) in high molecular weight (HMW, >10 kDa), medium molecular weight (MMW, 1-10 kDa), and low molecular weight (LMW, <1 kDa) gastrointestinal digests of glyoxal-glycated casein in the presence of CM. Fluorescent MRPs showed high stability, whereas UV-absorbent MRPs may be partially decomposed. A higher depletion rate of CML was found in the LMW fraction (38.7%) than in the MMW (21.7%) and HMW (9.6%) fractions. The 16S rRNA sequencing results revealed both beneficial and detrimental changes in CM composition induced by the glycated fractions. Generation of short-chain and branched-chain fatty acids in fermentation solutions with glycated fractions was significantly suppressed compared with that in fermentation solution with unglycated digests. This work revealed the possible interplay between peptide-bound MRPs and CM.

Site-specific chemoproteomic profiling of targets of glyoxal.

Aim: Advanced glycation end products (AGE) are the biomarkers of aging and diabetes which are formed via reactions between glycating agents and biomacromolecules. However, no proteomic study has been reported to systematically investigate the protein substrates of AGEs. Results: In this paper, we used an aniline-based probe to capture the glyoxal-imine intermediate which is the transition sate of glyoxal-derived AGEs. Combined with the tandem orthogonal proteolysis activity-based protein profiling strategy, we successfully identified 962 lysines modified by glyoxal. Conclusion: Enzymes in glycolysis are heavily modified by glyoxal and our biochemical experiments showed that glyoxal can significantly inhibit the activity of GAPDH and glycolysis. These data indicated that AGEs modifications may contribute to pathological processes through impairing the glycolytic process.

Study of Aberrant Modifications in Peptides as a Test Bench to Investigate the Immunological Response to Non-Enzymatic Glycation.

A side effect of diabetes is formation of glycated proteins and, from them, production of advanced early glycation end products that could determine aberrant immune responses at the systemic level. We investigated a relevant aberrant post-translational modification (PTM) in diabetes based on synthetic peptides modified on the lysine side chain residues with 1-deoxyfructopyranosyl moiety as a possible modification related to glycation. The PTM peptides were used as molecular probes for detection of possible specific autoantibodies developed by diabetic patients. The PDC-E2(167-186) sequence from the pyruvate dehydrogenase complex was selected and tested as a candidate peptide for antibody detection. The structure-based designed type I' ß-turn CSF114 peptide was also used as a synthetic scaffold. Twenty-seven consecutive type 1 diabetic patients and 29 healthy controls were recruited for the study. In principle, the 'chemical reverse approach', based on the use of patient sera to screen the synthetic modified peptides, leads to the identification of specific probes able to characterize highly specific autoantibodies as disease biomarkers of autoimmune disorders. Quite surprisingly, both peptides modified with the (1-deoxyfructosyl)-lysine did not lead to significant results. Both IgG and IgM differences between the two populations were not significant. These data can be rationalized considering that i) IgGs in diabetic subjects exhibit a high degree of glycation, leading to decreased functionality; ii) IgGs in diabetic subjects exhibit a privileged response vs proteins containing advanced glycation products (e.g., methylglyoxal, glyoxal, glucosone, hydroimidazolone, dihydroxyimidazolidine) and only a minor one with respect to (1-deoxyfructosyl)-lysine.

Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation.

To understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes with long acquisition times and high phototoxicity. Therefore, physiological live cell imaging is often unfeasible and chemical fixation is employed. Yet, fixation methods have not been rigorously investigated, in terms of pattern preservation, at the resolution at which cells can now be imaged. A key parameter for this is the time required until fixation is complete. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Other small aldehydes, glyoxal and acrolein, did not perform better. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from cells. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely absent with supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

Investigating the Glycating Effects of Glucose, Glyoxal and Methylglyoxal on Human Sperm.

Glycation is the non-enzymatic reaction between reducing sugars, such as glucose, and proteins, lipids or nucleic acids, producing Advanced Glycation End (AGE) products. AGEs, produced during natural senescence as well as through lifestyle factors such as diet and smoking, are key pathogenic compounds in the initiation and progression of diabetes. Importantly, many of these factors and conditions also have influence on male fertility, affecting sperm count and semen quality, contributing to the decreasing trend in male fertility. This study investigated the impact of AGEs on sperm damage. In vitro sperm glycation assays were used to determine the levels and localization of the potent AGE compound, carboxymethyl-lysine (CML) in response to treatment with the glycating compounds glucose, glyoxal and methylglyoxal. Sperm function assays were then used to assess the effects of glycation on motility and hyaluronan binding, and levels of oxidative DNA damage were analyzed through measurement of the marker, 8-oxoguanine. Results showed that glyoxal, but not glucose or methylglyoxal, induced significant increases in CML levels on sperm and this correlated with an increase in 8-oxoguanine. Immunocytochemistry revealed that AGEs were located on all parts of the sperm cell and most prominently on the head region. Sperm motility and hyaluronidase activity were not adversely affected by glycation. Together, the observed detrimental effects of the increased levels of AGE on DNA integrity, without an effect on motility and hyaluronidase activity, suggest that sperm may retain some fertilizing capacity under these adverse conditions.

Further characterization of the Maillard deglycase DJ-1 and its prokaryotic homologs, deglycase 1/Hsp31, deglycase 2/YhbO, and deglycase 3/YajL.

We reported recently that the Parkinsonism-associated protein DJ-1 and its bacterial homologs Hsp31, YhbO and YajL function as deglycases that repair proteins and nucleotides from endogeneous glycation by glyoxal and methylglyoxal, two reactive by-products of glucose metabolism responsible for up to 60% of glycation damage. Here, we show that DJ-1, deglycase 1 and deglycase 2 repair glyoxal- and methylglyoxal-glycated substrates, whereas deglycase 3 principally repairs glyoxal-glycated substrates. Moreover, deglycase 1 and 2 are overexpressed in stationary phase, whereas deglycase 3 is steadily expressed throughout bacterial growth. Finally, deglycase mutants overexpress glyoxalases, aldoketoreductases, glutathione-S-transferase and efflux pumps to alleviate carbonyl stress. In the discussion, we present an overview of the multiple functions of DJ-1 proteins. Our thourough work on deglycases provides compelling evidence that their previously reported glyoxalase III activity merely reflects their deglycase activity. Moreover, for their deglycase activity the Maillard deglycases likely recruit: i) their chaperone activity to interact with glycated proteins, ii) glyoxalase 1 activity to catalyze the rearrangement of Maillard products (aminocarbinols and hemithioacetals) into amides and thioesters, respectively, iii) their protease activity to cleave amide bonds of glycated arginine, lysine and guanine, and iv) glyoxalase 2 activity to cleave thioester bonds of glycated cysteine. Finally, because glycation affects many cellular processes, the discovery of the Maillard deglycases, awaited since 1912, likely constitutes a major advance for medical research, including ageing, cancer, atherosclerosis, neurodegenerative, post-diabetic, renal and autoimmune diseases.

Modification of chickpea cystatin by reactive dicarbonyl species: Glycation, oxidation and aggregation.

Reactive dicarbonyl species such as methylglyoxal (MGO) and glyoxal (GO) have recently received extensive attention due to their high reactivity and ability to modify biological substances such as proteins, phospholipids, and DNA. In case of proteins these reactive species mainly react with lysine and arginine residues to form AGEs, oxidative products, and aggregates. Chickpea cystatin (CPC) was incubated with varying concentrations of glyoxal and methylglyoxal which caused, along with altered secondary and tertiary structures, glycation, functional inactivation, altered redox state, cross-linking and high-molecular-mass aggregation. All these processes were examined and characterized by UV-Vis, fluorescence, and CD spectroscopies. Further characterization of CPC modified by reactive dicarbonyls was done by polyacrylamide gel electrophoresis which also showed alterations in the CPC molecules. Thus, in addition to describing the effects of GO and MGO on structure, conformation and function of CPC, this study also shows the relatively superior modifying effect of methylglyoxal for CPC in terms of glycation, oxidation and aggregation. This model system could shed some more light on the role of the reactive dicarbonyls in the specific alterations of proteins with different biological consequences having implications to ageing and disease such as diabetes.

Metformin attenuates myocardium dicarbonyl stress induced by chronic hypertriglyceridemia.

Reactive dicarbonyls stimulate production of advanced glycation endproducts, increase oxidative stress and inflammation and contribute to the development of vascular complications. We measured concentrations of dicarbonyls - methylglyoxal (MG), glyoxal (GL) and 3-deoxyglucosone (3-DG) - in the heart and kidney of a model of metabolic syndrome - hereditary hypertriglyceridemic rats (HHTg) and explored its modulation by metformin. Adult HHTg rats were fed a standard diet with or without metformin (300 mg/kg b.w.) and dicarbonyl levels and metabolic parameters were measured. HHTg rats had markedly elevated serum levels of triacylglycerols (p<0.001), FFA (p<0.01) and hepatic triacylglycerols (p<0.001) along with increased concentrations of reactive dicarbonyls in myocardium (MG: p<0.001; GL: p<0.01; 3-DG: p<0.01) and kidney cortex (MG: p<0.01). Metformin treatment significantly reduced reactive dicarbonyls in the myocardium (MG: p<0.05, GL: p<0.05, 3-DG: p<0.01) along with increase of myocardial concentrations of reduced glutathione (p<0.01) and glyoxalase 1 mRNA expression (p<0.05). Metformin did not have any significant effect on dicarbonyls, glutathione or on glyoxalase 1 expression in kidney cortex. Chronically elevated hypertriglyceridemia was associated with increased levels of dicarbonyls in heart and kidney. Beneficial effects of metformin on reactive dicarbonyls and glyoxalase in the heart could contribute to its cardioprotective effects.

Food-derived 1,2-dicarbonyl compounds and their role in diseases.

Reactive 1,2-dicarbonyl compounds (DCs) are generated from carbohydrates during food processing and storage and under physiological conditions. In the recent decades, much knowledge has been gained concerning the chemical formation pathways and the role of DCs in food and physiological systems. DCs are formed mainly by dehydration and redox reactions and have a strong impact on the palatability of food, because they participate in aroma and color formation. However, they are precursors of advanced glycation end products (AGEs), and cytotoxic effects of several DCs have been reported. The most abundant DCs in food are 3-deoxyglucosone, 3-deoxygalactosone, and glucosone, predominating over methylglyoxal, glyoxal, and 3,4-dideoxyglucosone-3-ene. The availability for absorption of individual DCs is influenced by the release from the food matrix during digestion and by their reactivity towards constituents of intestinal fluids. Some recent works suggest formation of DCs from dietary sugars after their absorption, and others indicate that certain food constituents may scavenge endogenously formed DCs. First works on the interplay between dietary DCs and diseases reveal an ambiguous role of the compounds. Cancer-promoting but also anticancer effects were ascribed to methylglyoxal. Further work is still needed to elucidate the reactions of DCs during intestinal digestion and pathophysiological effects of dietary DCs at doses taken up with food and in "real" food matrices in disease states such as diabetes, uremia, and cancer.

Protein Repair from Glycation by Glyoxals by the DJ-1 Family Maillard Deglycases.

DJ-1 and its prokaryotic homologs, Hsp31, YhbO and YajL from Escherichia coli and PfpI from Pyrococcus furiosus, repair proteins from glycation by glyoxals (R-CO-CHO), which constitute their major glycating agents. Glycation is a non-enzymatic covalent reaction discovered by Louis Camille Maillard in 1912, between reactive carbonyls (reducing sugars and glyoxals) and amino acids (cysteine, arginine and lysine), which inactivates proteins. By degrading Maillard adducts formed between carbonyls and thiols or amino groups, the DJ-1 family Maillard deglycases prevent the formation of the so-called advanced glycation end products (AGEs) that arise from Maillard adducts after dehydrations, oxidations and rearrangements. Since glycation is involved in ageing, cancer, atherosclerosis and cataracts, as well as post-diabetic, neurovegetatives and renal and autoimmune diseases, the DJ-1 deglycases are likely to play an important role in preventing these diseases. These deglycases, especially those from thermophilic organisms, may also be used to prevent the formation of dietary AGEs during food processing, sterilization and storage. They also prevent acrylamide formation in food, likely by degrading the asparagine/glyoxal Maillard adducts responsible for its formation. Since Maillard adducts are the substrates of the DJ-1 family deglycases, we propose renaming them Maillard deglycases.