Non-enzymatic Modification Of Proteins Research Articles

Analysis of Advanced Glycation Endproducts in Rat Tail Collagen and Correlation to Tendon Stiffening.

Methylglyoxal is a major 1,2-dicarbonyl compound in vivo and leads to nonenzymatic protein modifications, known as advanced glycation endproducts. Especially long-lived proteins like collagen are prone to changes of the mechanical or biological function, respectively, by accumulation of Maillard-derived modifications. Specifically, the resulting nonenzymatic cross-link structures in parallel to the natural maturation process of collagen fibrils lead to complications with age or during disease. A novel lysine-lysine amide cross-link derived from methylglyoxal, 2,15-diamino-8-methyl-9-oxo-7,10-diaza-1,16-hexadecanedioic acid, named MOLA, was synthesized and identified in vitro and in vivo. Tail tendons of young, adult, and old rats (3, 12, and 22 months) were enzymatically digested prior to analysis of acid-labile glycation products via liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a result, nine monovalent amino acid modifications, mostly originating from methylglyoxal (36 μmol/mol leucine-equivalents in total), and four glycation cross-links (0.72 μmol/mol glucosepane, 0.24 μmol/mol DODIC (3-deoxyglucosone-derived imidazoline cross-link), 0.04 μmol/mol MODIC (methylglyoxal-derived imidazoline cross-link), 0.34 μmol/mol MOLA) were quantitated in senescent tendon collagen. The results correlated with increased tail tendon breaking time from 10 to 190 min and indicate that methylglyoxal is a major player in the aging process of connective tissue.

Epigenetic Regulation of Gene Transcription by Nonenzymatic Protein Modification

Epigenetic Regulation of Gene Transcription by Nonenzymatic Protein Modification

Protein Carbamylation in Chronic Kidney Disease and Dialysis.

Protein carbamylation is a nonenzymatic posttranslational protein modification that can be driven, in part, by exposure to urea's dissociation product, cyanate. In humans, when kidney function is impaired and urea accumulates, systemic protein carbamylation levels increase. Additional mediators of protein carbamylation have been identified including inflammation, diet, smoking, circulating free amino acid levels, and environmental exposures. Carbamylation reactions on proteins are capable of irreversibly changing protein charge, structure, and function, resulting in pathologic molecular and cellular responses. Carbamylation has been mechanistically linked to the biochemical pathways implicated in atherosclerosis, dysfunctional erythropoiesis, kidney fibrosis, autoimmunity, and other pathological domains highly relevant to patients with chronic kidney disease. In this review, we describe the biochemical impact of carbamylation on human proteins, the mechanistic role carbamylation can have on clinical outcomes in kidney disease, the clinical association studies of carbamylation in chronic kidney disease, including patients on dialysis, and the promise of therapies aimed at reducing carbamylation burden in this vulnerable patient population.

Increased serotransferrin and ceruloplasmin turnover in diet-controlled patients with type 2 diabetes

Type 2 diabetes mellitus (T2DM) is associated with oxidative stress and perturbed iron metabolism. Serotransferrin (Trf) and ceruloplasmin (Cp) are two key proteins involved in iron metabolism and anti-oxidant defense. Non-enzymatic glycation and oxidative modification of plasma proteins are known to occur under hyperglycemia and oxidative stress. In this study, shotgun proteomics and 2H2O-based metabolic labeling were used to characterize post-translational modifications and assess the kinetics of Trf and Cp in T2DM patients and matched controls in vivo. Six early lysine (Amadori) and one advanced arginine glycation were detected in Trf. No glycation, but five asparagine deamidations, were found in Cp. T2DM patients had increased fractional catabolic rates of both Trf and Cp that correlated with HbA1c (p < 0.05). The glycated Trf population was subject to an even faster degradation compared to the total Trf pool, suggesting that hyperglycemia contributed to an increased Trf degradation in T2DM patients. Enhanced production of Trf and Cp kept their levels stable. The changes in Trf and Cp turnover were associated with increased systemic oxidative stress without any alteration in iron status in T2DM. These findings can help better understand the potential role of altered Trf and Cp metabolism in the pathogenesis of T2DM and other diseases.

The role of glycation in the pathogenesis of aging and its prevention through herbal products and physical exercise.

[Purpose]Advanced glycation end products (AGEs) are non-enzymatic modifications of proteins or lipids after exposure to sugars. In this review, the glycation process and AGEs are introduced, and the harmful effects of AGEs in the aging process are discussed.[Methods]Results from human and animal studies examining the mechanisms and effects of AGEs are considered. In addition, publications addressing means to attenuate glycation stress through AGE inhibitors or physical exercise are reviewed.[Results]AGEs form in hyperglycemic conditions and/or the natural process of aging. Numerous publications have demonstrated acceleration of the aging process by AGEs. Exogenous AGEs in dietary foods also trigger organ dysfunction and tissue aging. Various herbal supplements or regular physical exercise have beneficial effects on glycemic control and oxidative stress with a consequent reduction of AGE accumulation during aging.[Conclusion]The inhibition of AGE formation and accumulation in tissues can lead to an increase in lifespan.

A Perspective on Reagent Diversity and Non-covalent Binding of Reactive Carbonyl Species (RCS) and Effector Reagents in Non-enzymatic Glycation (NEG): Mechanistic Considerations and Implications for Future Research.

This perspective focuses on illustrating the underappreciated connections between reactive carbonyl species (RCS), initial binding in the nonenzymatic glycation (NEG) process, and nonenzymatic covalent protein modification (here termed NECPM). While glucose is the central species involved in NEG, recent studies indicate that the initially-bound glucose species in the NEG of human hemoglobin (HbA) and human serum albumin (HSA) are non-RCS ring-closed isomers. The ring-opened glucose, an RCS structure that reacts in the NEG process, is most likely generated from previously-bound ring-closed isomers undergoing concerted acid/base reactions while bound to protein. The generation of the glucose RCS can involve concomitantly-bound physiological species (e.g., inorganic phosphate, water, etc.); here termed effector reagents. Extant NEG schemes do not account for these recent findings. In addition, effector reagent reactions with glucose in the serum and erythrocyte cytosol can generate RCS (e.g., glyoxal, glyceraldehyde, etc.). Recent research has shown that these RCS covalently modify proteins in vivo via NECPM mechanisms. A general scheme that reflects both the reagent and mechanistic diversity that can lead to NEG and NECPM is presented here. A perspective that accounts for the relationships between RCS, NEG, and NECPM can facilitate the understanding of site selectivity, may help explain overall glycation rates, and may have implications for the clinical assessment/control of diabetes mellitus. In view of this perspective, concentrations of ribose, fructose, Pi, bicarbonate, counter ions, and the resulting RCS generated within intracellular and extracellular compartments may be of importance and of clinical relevance. Future research is also proposed.

Aldo-ketoreductase 1 (AKR1) improves seed longevity in tobacco and rice by detoxifying reactive cytotoxic compounds generated during ageing

BackgroundMaintenance of seed viability is an important factor for seedling vigour and plant establishment. Lipid peroxidation mediated reactive carbonyl compounds (RCC’s) and non-enzymatic modifications of proteins through Maillard and Amadori products reduce seed viability and seedling vigour.ResultsIn this study, the relevance of RCCs on genotypic variation in rice seed viability and overexpression of an aldo-ketoreductase (AKR1) enzyme that detoxify cytotoxic compounds to improve seed viability and vigour was studied. Physiological and biochemical approaches were integrated to quantify the variation in seed viability and seedling vigour in rice genotypes after exposing to ageing treatment. AKR1 was overexpressed in a susceptible rice genotype and tobacco to study the relevance of reduced RCC’s on seed viability and seedling vigour. The glycation and lipid peroxidation compounds accumulated after accelerated ageing treatments in rice genotypes. The accumulation of malondialdehyde, methyl glyoxal, Maillard and Amadori products affected the seed viability and germination as they showed a significant negative relationship. The transgenic rice and tobacco seeds expressing AKR1 showed lower levels of cytotoxic compounds and glycation products that resulted in improved seed viability and seedling vigour in rice and tobacco.ConclusionsThe study demonstrates that, reactive cytotoxic compounds affect the seed viability during storage. Detoxification of reactive cytotoxic compounds by Aldo-keto reductases is one of the mechanisms to improve the seed longevity during storage.

Elevation of Fumarate Levels Compromise Redox Control and Viability in Mycobacterium tuberculosis

In this issue of Cell Chemical Biology, Ruecker etal. (2017) show that fumarase depletion in Mycobacterium tuberculosis leads to fumarate, a TCA cycle intermediate, accumulation, causing succination of a range of thiol-containing metabolites and proteins. Fumarate is bactericidal to the pathogen, and its accumulation may enhance the bactericidal effector mechanisms of other TCA cycle intermediates that accumulate due to activation of infected macrophages.

A Prob(e)able Route to Lysine Acylation

Non-enzymatic modification of proteins by acyl-CoA species involved in intermediary metabolism is a possible explanation for widespread protein acylation. In this issue, Kulkarni etal. (2017) develop a set of chemoproteomic probes to interrogate the role of malonyl-CoA in mediating protein malonylation and find malonylation influences glycolysis in cancer cells.

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling

Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.

Phagocytosis of Advanced Glycation End Products (AGEs) in Macrophages Induces Cell Apoptosis.

Advanced glycation end products (AGEs) are the products of a series of nonenzymatic modifications of proteins by reducing sugars. AGEs play a pivotal role in development of diabetic complications and atherosclerosis. Accumulation of AGEs in a vessel wall may contribute to the development of vascular lesions. Although AGEs have a diverse range of bioactivities, the clearance process of AGEs from the extracellular space, including the incorporation of AGEs into specific cells, subcellular localization, and the fate of AGEs, remains unclear. In the present study, we examined the kinetics of the uptake of AGEs by mouse macrophage J774.1 cells in vitro and characterized the process. We demonstrated that AGEs bound to the surface of the cells and were also incorporated into the cytoplasm. The temperature- and time-dependent uptake of AGEs was saturable with AGE concentration and was inhibited by cytochalasin D but not chlorpromazine. We also observed the granule-like appearance of AGE immunoreactivity in subcellular localizations in macrophages. Higher concentrations of AGEs induced intracellular ROS and 4-HNE, which were associated with activation of the NF-κB pathway and caspase-3. These results suggest that incorporation of AGEs occurred actively by endocytosis in macrophages, leading to apoptosis of these cells through NF-κB activation.

The possible impact of advanced glycation end products on pregnancy outcome in women with diabetes mellitus type 1.

Diabetes mellitus (DM) is a group of metabolic disorders of carbohydrate metabolism in which glucose is underutilized, resulting in hyperglycemia. Reproductive impairment in poorly controlled diabetes mellitus type 1 (T1DM) results from a combined effect of insulin deficiency and hyperglycemia that disrupt the functioning of metabolic signals participating in the regulation of the reproductive system. Good metabolic control as a result of intensive insulin therapy has a great impact on the fertility and childbearing possibilities in the T1DM females. Advanced glycation end products (AGEs) are formed by nonenzymatic modification of proteins, lipids, and nucleic acids by glucose. The formation and accumulation of AGEs are known to progress at an accelerated rate in diabetes. AGEs either act on the pro-inflammatory cell surface receptors called RAGE or bind to the circulating anti-inflammatory sRAGE that prevents activation of cell-surface RAGE by AGEs and other proinflammatory ligands. Pregnancy has been found to induce a significant increase in RAGE protein levels in both myometrium and omental vasculature. This review will focus on the role of AGEs and RAGE in pregnancy complicated by DM type 1 as well as ways to reduce the rate of congenital malformations in the offspring of diabetic type 1 women.

Preserving Brain Function in Aging: The Anti-glycative Potential of Berry Fruit.

Advanced glycation end products (AGEs) are naturally occurring macromolecules that are formed in vivo by the non-enzymatic modification of proteins, lipids, or nucleic acids by sugar, even in the absence of hyperglycemia. In the diet, AGEs are found in animal products, and additional AGEs are produced when those foods are cooked at high temperatures. Studies have linked AGEs to various age-related physiological changes, including wrinkles, diabetic complications, and neurodegenerative disease, including Alzheimer's disease. Dietary berry fruits have been shown to reduce the severity or slow the progression of many physiological changes and disease pathologies that accompany aging. Emerging evidence has shown that the phytochemicals found in berry fruits exhibit anti-glycative activity. In this review, we briefly summarize the current evidence supporting the neuroprotective anti-glycative activity of berry fruits and their potential to preserve cognitive function during aging.

Determination of advanced glycation end-products and antibodies against them (anti-CML and anti-CEL) in the serum of Graves' orbitopathy patients before and after methylprednisolone treatment.

The glycation process is a non-enzymatic modification of proteins occurring due to the reactions of reductive carbohydrates. The glycated residues lose their biological functions, and their removal process is ineffective. They accumulate, and as a result they cause an immunological response. The aim of this study was a determination of the concentrations of advanced glycation end-products and antibodies against carboxymethyl lysine (anti-CML) and carboxyethyl lysine (anti-CEL) in the sera of Graves' orbitopathy patients. The study group were patients from the Division of Endocrinology of the Medical University of Silesia (n = 25) suffering from Graves' orbitopathy. The concentration of AGE-peptides using flow spectrofluorimetry method, and anti-CML and anti-CEL IgG antibodies using immunoenzymatic technique (ELISA), were measured in patients sera before and after methylprednisolone treatment. In sera of the study group the concentrations of AGE-peptides and anti-CML were significantly lower before and after treatment in comparison to the control group (p < 0.05). Mean values of anti-CEL concentrations were comparable (at both phases of treatment) with the value observed in the control group. After treatment the concentrations of AGE-peptides and anti-CEL significantly decreased (p < 0.05); however, the concentration of anti-CML was also lower but the observed change was not significant (p > 0.05). In the course of Graves' orbitopathy the glycation process is disturbed. The treatment modifies significantly the process by lowering the concentration of advanced glycation end-products and suppressing the immune response to them. (Endokrynol Pol 2016; 67 (4): 383-389).

Relationship of Advanced Glycation End Products With Cardiovascular Disease in Menopausal Women

Cardiovascular disease (CVD) represents the most significant cause of death in postmenopausal women. Advanced glycation end products (AGEs) are formed by nonenzymatic modification of proteins, lipids, and nucleic acids by glucose. This review focuses on the contribution of AGEs and their receptors to the development of CVD in menopause. Advanced glycation end products circulate and activate the proinflammatory endothelial cell surface receptor called RAGE, bind to the extracellular matrix of the cardiovascular system, or bind to the circulating anti-inflammatory soluble form of RAGE (sRAGE). Data emerging from human and animal studies suggest that AGEs and both receptors (RAGE and sRAGE) are implicated in the pathophysiology of CVD. Particular emphasis has been given to the role of AGE-RAGE axis in oxidative stress, inflammation, endothelial cell toxicity, and progression of atherosclerosis in menopause. Data accruing from human and animal studies suggest that RAGE expression level and circulating sRAGE level are associated with estradiol and are correlated with CVD risk factors, such as adiposity, dyslipidemia, insulin resistance, diabetes, and metabolic syndrome. By recognizing the impact of AGEs on atherosclerosis, pharmacological strategies targeting the AGE-RAGE pathway hold therapeutic potential for CVD in menopausal women.

Advanced glycation endproducts interfere with adhesion and neurite outgrowth.

Advanced glycation endproducts (AGEs) represent a non-enzymatic posttranslational protein modification. AGEs are generated by a series of chemical reactions of free reducing monosaccharides, such as glucose, fructose or metabolites of the monosaccharide metabolism with amino groups of proteins. After oxidation, dehydration and condensation, stable AGE-modifications are formed. AGE-modified proteins accumulate in all cells and tissues as a normal feature of ageing and correlate with the glucose concentration in the blood. AGEs are increased in diabetic patients and play a significant role in the pathogenesis of most age-related neural disorders, such as Alzheimer’s disease. We examined the role of AGEs on neurite outgrowth of PC12 cells. We induced the formation of AGEs using the reactive carbonyl compound methylglyoxal (MGO) as a physiological metabolite of glucose. We found that AGE-modification of laminin or collagen interfered with adhesion but not with neurite outgrowth of PC12 cells. Furthermore, the AGE-modification of PC12 cell proteins reduced NGF-induced neurite outgrowth. In conclusion, our data show that AGEs negatively influence neural plasticity.

Reprint of “Accumulation of modified proteins and aggregate formation in aging”

Increasing cellular damage during the aging process is considered to be one factor limiting the lifespan of organisms. Besides the DNA and lipids, proteins are frequent targets of non-enzymatic modifications by reactive substances including oxidants and glycating agents. Non-enzymatic protein modifications may alter the protein structure often leading to impaired functionality. Although proteolytic systems ensure the removal of modified proteins, the activity of these proteases was shown to decline during the aging process. The additional age-related increase of reactive compounds as a result of impaired antioxidant systems leads to the accumulation of damaged proteins and the formation of protein aggregates. Both, non-enzymatic modified proteins and protein aggregates impair cellular functions and tissue properties by a variety of mechanisms. This is increasingly important in aging and age-related diseases. In this review, we will give an overview on oxidation and glycation of proteins and the function of modified proteins in aggregate formation. Furthermore, their effects as well as their role in aging and age-related diseases will be highlighted.

Protein Carbamylation in Kidney Disease: Pathogenesis and Clinical Implications

Carbamylation describes a nonenzymatic posttranslational protein modification mediated by cyanate, a dissociation product of urea. When kidney function declines and urea accumulates, the burden of carbamylation naturally increases. Free amino acids may protect proteins from carbamylation, and protein carbamylation has been shown to increase in uremic patients with amino acid deficiencies. Carbamylation reactions are capable of altering the structure and functional properties of certain proteins and have been implicated directly in the underlying mechanisms of various disease conditions. A broad range of studies has demonstrated how the irreversible binding of urea-derived cyanate to proteins in the human body causes inappropriate cellular responses leading to adverse outcomes such as accelerated atherosclerosis and inflammation. Given carbamylation's relationship to urea and the evidence that it contributes to disease pathogenesis, measurements of carbamylated proteins may serve as useful quantitative biomarkers of time-averaged urea concentrations while also offering risk assessment in patients with kidney disease. Moreover, the link between carbamylated proteins and disease pathophysiology creates an enticing therapeutic target for reducing the rate of carbamylation. This article reviews the biochemistry of the carbamylation reaction, its role in specific diseases, and the potential diagnostic and therapeutic implications of these findings based on recent advances.

Prince Cangrande’s Collagen: Study of Protein Modification on the Mummy of the Lord of Verona, Italy (1291–1329 AD)

The natural mummy of prince Cangrande, Lord of Verona, Italy (1291–1329 AD) was studied. Two samples were taken: rib bone and muscle. These samples were cleaved with trypsin and analysed by liquid chromatographic methods coupled to mass spectrometry (Q-TOF, ion-trap). Special attention was devoted to nonenzymatic protein modification––the deamidation of asparagine and glutamine. A huge amount of collagen was determined in the tissues of the mummy (covering over 80 % of the sequence)––collagen type I was identified in the rib bone and collagen types I and III in the muscle. A high overall percentage of asparaginyl and glutaminyl residues were deamidated (up to 92 %). In agreement with the literature we can suppose that the deamidation of really old samples (at least 100-years-old) is mainly dependent on the burial conditions and/or thermal age and cannot serve as a precise “molecular clock”.

Accumulation of modified proteins and aggregate formation in aging

Increasing cellular damage during the aging process is considered to be one factor limiting the lifespan of organisms. Besides the DNA and lipids, proteins are frequent targets of non-enzymatic modifications by reactive substances including oxidants and glycating agents. Non-enzymatic protein modifications may alter the protein structure often leading to impaired functionality. Although proteolytic systems ensure the removal of modified proteins, the activity of these proteases was shown to decline during the aging process. The additional age-related increase of reactive compounds as a result of impaired antioxidant systems leads to the accumulation of damaged proteins and the formation of protein aggregates. Both, non-enzymatic modified proteins and protein aggregates impair cellular functions and tissue properties by a variety of mechanisms. This is increasingly important in aging and age-related diseases. In this review, we will give an overview on oxidation and glycation of proteins and the function of modified proteins in aggregate formation. Furthermore, their effects as well as their role in aging and age-related diseases will be highlighted.