Formation Of Protein Crosslinks Research Articles

The cysteine residue in beta-lactoglobulin reacts with oxidized tyrosine residues in beta-casein to give casein-lactoglobulin dimers

Proteins are modified during milk processing and storage, with sidechain oxidation and crosslinking being major consequences. Despite the prevalence and importance of proteins in milk, and particularly caseins (∼80% of total content), the nature of the cross-links formed by oxidation, and their mechanisms of formation, are poorly characterized. In this study, we investigated the formation and stability of cross-links generated by the nucleophilic addition of Cys residues to quinones generated on oxidation of Tyr residues. The mechanisms and stability of these adducts was explored using ubiquitin as a model protein, and β-casein. Ubiquitin and β-casein were oxidized using a rose Bengal/visible light/O2 system, or by the enzyme tyrosinase. The oxidized proteins were incubated with glutathione or β-lactoglobulin (non-oxidized, but unfolded by treatment at 70 °C), before analysis by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that Cys-quinone adducts are readily-formed, and are stable for >48 h. Thus, oxidized β-casein reacts efficiently with the thermally unfolded β-lactoglobulin, likely via Michael addition of the exposed Cys to a Tyr-derived quinone. These data provide a novel, and possibly general, mechanism of protein cross-link formation, and provides information of the stability of these species that have potential as markers of protein quality.

Evaluation of Major Constituents of Medicinally Important Plants for Anti-Inflammatory, Antidiabetic and AGEs Inhibiting Properties: In Vitro and Simulatory Evidence.

Diabetes mellitus (DM) is a global health concern that is associated with several micro- and macrovascular complications. We evaluated several important medicinal plant constituents, including polyphenols and flavonoids, for α-glucosidase inhibition, AGEs’ inhibitory activities using oxidative and no-oxidative assays, the inhibition of protein cross link formation, 15-lipoxydenase inhibition and molecular docking. The molecular docking studies showed high binding energies of flavonoids for transcriptional regulars 1IK3, 3TOP and 4F5S. In the α-glucosidase inhibition assay, a significant inhibition was noted for quercitrin (IC50 7.6 µg/mL) and gallic acid (IC50 8.2 µg/mL). In the AGEs inhibition assays, quercetin showed significant results in both non-oxidative and (IC50 0.04 mg/mL) and oxidative assays (IC50 0.051 mg/mL). Furthermore, quercitrin showed inhibitory activity in the non-oxidative (IC50 0.05 mg/mL) and oxidative assays (IC50 0.34 mg/mL). A significant inhibition of protein cross link formation was observed by SDS-PAGE analysis. Quercitrin (65%) and quercetin (62%) showed significant inhibition of 15-lipoxygenase. It was thus concluded that flavonoids and other polyphenols present in plant extracts can be effective in management of diabetes and allied co-morbidities.

Computational analysis and in vitro investigation on Citrus flavonoids for inflammatory, diabetic and AGEs targets

Flavonoids are a diverse class of polyphenolic substances largely found in plants including citrus peels and are reported to posess a variety of biological activities. We investigated important flavonoids apigenin, hesperidin, narigin, quercetin and tangeritine against diabetes and associated conditions. In current project drug likeness, ADMET analysis, molecular docking and in vitro assays were performed. The apigenin, quercetin and tanagretin exhibited compliance with Lipinski’s rule of five. The molecular docking analysis showed best fit in transcriptional regulator 3TOP and 1IK3 in all tested compounds. During antioxidant assays, all flavonoids presented excellent activities. In the α-glucosidase assay, quercetin showed highest inhibition (76% at final concentration of 52 µg/ml) followed by tangeritin (73% at final concentration of 52 µg/ml). In case of 15-Lox assay, highest inhibition was seen in case of quercetin (75%) followed by apigenin (53%). In the AGEs assay, the quercetin showed 47% inhbition of protein cross link formation preceeded by the tenegretin exhited 37% inhibition. It was therefore concluded that tested flavonoids have significant activities in both in silico and in vitro models that is mainly due to differences in structural features and polar surface area.

Suppression of glycated protein cross-link formation and cross-link cleavage reaction of edible purple Chrysanthemum flower extract

Suppression of glycated protein cross-link formation and cross-link cleavage reaction of edible purple Chrysanthemum flower extract

Protein cross-link formation by oxidation of disulfide bonds

Protein cross-link formation by oxidation of disulfide bonds

Formation of protein cross-links by singlet oxygen-mediated disulfide oxidation

Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen (1O2)-mediated oxidation of multiple proteins (α-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDS-PAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the 1O2 concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with 18O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta-2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide ‘proof-of-concept’ for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies.

The use of microbial transglutaminase in a bread system: A study of gluten protein structure, deamidation state and protein digestion

Microbial transglutaminase (mTG) catalyses the formation of protein crosslinks, deamidating glutamine in a side-reaction. Gluten deamidation by human tissue transglutaminase is critical to activate celiac disease pathogenesis making the addition of mTG to wheat-based products controversial. The ability of mTG (0–2000 U.kg−1) to alter gluten’s structure, digestibility and the deamidation state of six immunogenic gluten peptides within bread was investigated. Gluten’s structure was altered when mTG exceeded 100 U.kg−1, determined by confocal microscopy, extractability and free sulfhydryl assays. The effect of mTG on six immunogenic peptides was investigated by in vitro digestion (INFOGEST) and mass spectrometry. The addition of mTG to bread (0–2000 U.kg−1) did not alter the deamidation state or digestibility of the immunogenic peptides investigated. Overall, this investigation indicated that the addition of mTG to bread does not create activated gluten peptides. This analysis provides evidence for risk assessments of mTG as a food processing aid.

Alagebrium and Complications of Diabetes Mellitus.

Glycation is the process of linking a sugar and free amino groups of proteins. Cross-linking of glycation products to proteins results in the formation of cross-linked proteins that inhibit the normal functioning of the cell. Advanced glycation end products (AGEs) are risk molecules for the cell aging process. These ends products are increasingly synthesized in diabetes and are essentially responsible for diabetic complications. They accumulate in the extracellular matrix and bind to receptors (receptor of AGE [RAGE]) to generate oxidative stress and inflammation. particularly in the cardiovascular system. Treatment methods targeting the AGE system may be of clinical importance in reducing and preventing the complications induced by AGEs in diabetes and old age. The AGE cross-link breaker alagebrium (a thiazolium derivative) is the most studied anti-AGE compound in the clinical field. Phase III clinical studies with alagebrium have been successfully conducted, and this molecule has positive effects on cardiovascular hypertrophy, diabetes, hypertension, vascular sclerotic pathologies, and similar processes. However, the mechanism is still not fully understood. The primary mechanism is that alagebrium removes newly formed AGEs by chemically separating α-dicarbonyl carbon-carbon bonds formed in cross-linked structures. However, it is also reported that alagebrium is a methylglyoxal effective inhibitor. It is not yet clear whether alagebrium inhibits copper-catalyzed ascorbic acid oxidation through metal chelation or destruction of the AGEs. It is not known whether alagebrium has a direct association with RAGEs. The safety profile is favorably in humans, and studies have been terminated due to financial insufficiency and inability to license as a drug.

Effects of malondialdehyde as a byproduct of lipid oxidation on protein oxidation in rabbit meat

The effects of malondialdehyde (MDA) as a byproduct of lipid oxidation on myoglobin and myofibrillar proteins (MP) oxidations in muscle homogenates containing components native to rabbit muscle were investigated. For myoglobin, MDA could lead to increase in metmyoglobin percentage. For MP, MDA could promote protein carbonylation and loss of tryptophan fluorescence. In addition, MDA could affect reactive oxygen species (ROS)-generating system by promoting the formation of hypervalent myoglobin species and release of non-heme iron. The result of MDA-MP adducts fluorescence intensity indicated that ROS-generating systems may be the main reason for protein carbonylation at the later of incubation treatment. SDS-PAGE analysis revealed that the ability of ROS-generating systems to facilitate protein oxidation was enhanced with MDA, which was responsible for the formation of protein cross-linking throughout incubation treatment. Taken together, the ability of MDA on promoting the oxidation of MP in rabbit muscle homogenates may be relied on both adduct reactions and the influence on ROS-generating system.

Molecular interactions and redox effects of carvacrol and thymol on myofibrillar proteins using a non-destructive and solvent-free methodological approach

The present study provides molecular insight into the effect of thymol and carvacrol on the oxidative damage caused to myofibrillar proteins by a hydroxyl-radical generating system (HRGS). An innovative model system was designed, in which gels, prepared with increasing levels of myofibrillar proteins, were oxidized by a HRGS (Fe3+/H2O2, 60 °C and 7 days) in the presence of lipids. The molecular affinity between myofibrillar proteins and both terpenes, as well as their effect on the oxidative stability of the gel systems, were studied using a non-destructive and solvent-free procedure based on fluorescence spectroscopy. Carvacrol displayed more affinity than thymol for establishing chemical interactions with protein residues. Both terpenes exhibited a significant antioxidant potential against the generation of lipid-derived volatile carbonyls and against the formation of protein crosslinking. This procedure may be applied to meat products to assess the effectiveness of a given antioxidant additive without size reduction or sample processing.

Underlying connections between the redox system imbalance, protein oxidation and impaired quality traits in pale, soft and exudative (PSE) poultry meat

The connections between the redox imbalance in post-mortem muscle, early protein oxidation and the onset of pale, soft and exudative (PSE) condition in chicken breast are studied. PSE was induced by incubation of post-mortem chicken carcasses at 37°C for 200min. PSE-induced muscle consistently had faster pH decline and lower pH at 200min (5.84 vs. 6.59) and 24h (5.69 vs. 5.96), higher L∗ (54.4 vs. 57.3), and lower texture and water holding capacity (WHC) than normal meat. The activities of catalase, glutathione peroxidase and superoxide dismutase were significantly lower in PSE-induced samples than in the normal counterparts. PSE was more susceptible to proteolysis and protein oxidation than normal meat during succeeding chilled storage with more intense tryptophan and thiols depletion, higher protein carbonylation and more intense formation of protein cross-links. We provide plausible explanations to support the role of protein oxidation in the impaired quality PSE chicken.

A Novel Mechanism by Which Tissue Transglutaminase Activates Signaling Events That Promote Cell Survival

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

Effect of high-oxygen atmosphere packaging on oxidative stability and sensory quality of two chicken muscles during chill storage

The oxidative stability and sensory quality of chicken breast (m. pectoralis) and thigh (m. peroneus longus) stored in high-oxygen modified atmosphere (MAP-O2), non-oxygen modified atmosphere (MAP-N2), or vacuum for up to 9 days at 5 °C were investigated. Protein thiol concentration in breasts and thighs decreased significantly between 5 and 9 days storage in MAP-O2 compared to non-oxygen storage, and resulted in myosin heavy chain disulfide cross-links. Thiol loss and protein cross-link formation were more pronounced in thighs compared to breasts, and a similar tendency was seen for the formation of secondary lipid oxidation products. However, while breast stored in MAP-O2 clearly scored lower in tenderness and higher in rancidity compared to breast in non-oxygen storage, the effect of MAP-O2 for the sensory quality of thigh was negligible. These results show that thigh is more suitable for storage in MAP-O2 than breast, indicating that the negative effect MAP-O2 may have on the oxidative stability and sensory quality of meat varies between different muscles.

Thiol oxidation and protein cross-link formation during chill storage of pork patties added essential oil of oregano, rosemary, or garlic

The effect of two levels (0.05% and 0.4%) of essential oil of rosemary, oregano, or garlic on protein oxidation in pork patties was studied during storage under modified atmosphere (MAP: 70% O2: 20% CO2: 10% N2) or under aerobic conditions (AE) at 4°C. The oxidative stability of the meat proteins was evaluated as loss of thiols for up to 9days of storage, and as formation of myosin cross-links analyzed by SDS-PAGE after 12days of storage. Protein thiols were lost during storage to yield myosin disulfide cross-links. Essential oils of rosemary and oregano were found to retard the loss of thiols otherwise resulting in myosin cross-links. Garlic essential oil, on the contrary, was found to promote protein oxidation, as seen by an extreme loss in thiol groups, and elevated myosin cross-link formation compared to control.

Effects of Hydrolysed Whey Proteins on the Techno-Functional Characteristics of Whey Protein-Based Films.

Pure whey protein isolate (WPI)-based cast films are very brittle due to its strong formation of protein cross-linking of disulphide bonding, hydrogen bonding as well as hydrophobic and electrostatic interactions. However, this strong cross-linking is the reason for its final barrier performance. To overcome film brittleness of whey protein layers, plasticisers like glycerol are used. It reduces intermolecular interactions, increases the mobility of polymer chains and thus film flexibility can be achieved. The objective of this study was to investigate the influence of hydrolysed whey protein isolate (WPI) in whey protein isolate-based cast films on their techno-functional properties. Due to the fact, that the addition of glycerol is necessary but at the same time increases the free volume in the film leading to higher oxygen and water vapour permeability, the glycerol concentration was kept constant. Cast films with different ratios of hydrolysed and not hydrolysed WPI were produced. They were characterised in order to determine the influence of the lower molecular weight caused by the addition of hydrolysed WPI on the techno-functional properties. This study showed that increasing hydrolysed WPI concentrations significantly change the mechanical properties while maintaining the oxygen and water vapour permeability. The tensile and elastic film properties decreased significantly by reducing the average molecular weight whereas the yellowish coloration and the surface tension considerably increased. This study provided new data which put researchers and material developers in a position to tailor the characteristics of whey protein based films according to their intended application and further processing.

Cross-Linking Behavior and Foaming Properties of Bovine α-Lactalbumin after Glycation with Various Saccharides

α-Lactalbumin was glycated via the Maillard reaction in the dry state using various mono- and oligosaccharides. The reaction resulted not only in coupling of the saccharides to α-lactalbumin but also in cross-linked proteins. The glycation rate and the extent of cross-link formation were highly dependent on the saccharide used. Glycation by arabinose and xylose led to a very fast protein cross-link formation, whereas glucose showed a relatively low protein cross-linking ability. The stability of foams, created using the various glycated protein samples, depended on the type of saccharide used, the extent of glycation, and possibly the amount of cross-linked protein. Compared to nonmodified α-lactalbumin, glycation with rhamnose and fucose improved foam stability, whereas application of glucose, galacturonic acid, and their oligosaccharides did not exert a clear effect. Mass spectrometric analysis revealed that dehydration of the Amadori products is an indicator of the formation of protein cross-links.

4-Methylcatechol Inhibits Protein Oxidation in Meat but Not Disulfide Formation

The interaction between phenolic compounds and protein thiols was investigated in minced beef with or without 500 ppm 4-methylcatechol (4-MC) that had been stored under oxygen or argon for 7 days (4 °C). Myofibrillar protein isolates were extracted, and the oxidative stability evaluated by the protein radical intensity measured by ESR spectroscopy was found to be improved by 4-MC as well as by storage without oxygen. Significant loss of thiols was found in samples stored under oxygen compared to argon, whereas an additional loss was found in samples with added 4-MC stored under oxygen. In beef with added 4-MC, LC-MS analysis showed formation of thiol-quinone adducts, which may explain the observed additional loss of thiols. Although storage without oxygen inhibited protein cross-link formation as evaluated by SDS-PAGE, both in presence and in the absence of 4-MC, no inhibitory effect of 4-MC was found on the formation of protein disulfide cross-links in beef stored under oxygen.

Thiol–Quinone Adduct Formation in Myofibrillar Proteins Detected by LC-MS

Protein oxidation in meat is considered to decrease meat tenderness due to protein disulfide cross-link formation of thiol-containing amino acid residues. An LC-MS method for detection of thiol-quinone adducts (RS-QH(2)) in myofibrillar proteins was developed to investigate the interaction between phenols, as protective antioxidants, and proteins from meat under oxidative conditions using aqueous solutions of (i) cysteine (Cys), (ii) glutathione (GSH), (iii) bovine serum albumin (BSA), or (iv) a myofibrillar protein isolate (MPI). The aqueous solutions were incubated at room temperature (30 min) with 4-methyl-1,2-benzoquinone (4MBQ) prepared from oxidation of 4-methylcatechol (4MC) by periodate resin or incubated at room temperature (5 h) with 4MC and Fe(II)/H(2)O(2). GSH, BSA, and MPI were hydrolyzed (6 N HCl, 110 °C, 22 h) after incubation, and the cysteine-quinone adduct, Cys-QH(2) (m/z 244.2) was identified according to UV and mass spectra after separation on an RP-C18 column. The thiol-quinone adduct was present in all thiol systems after incubation with 4MBQ or 4MC oxidized by Fe(II)/H(2)O(2). Direct reaction with 4MBQ resulted in each case in increased Cys-QH(2) formation compared to simultaneous oxidation of thiol source and 4MC with Fe(II)/H(2)O(2). The covalent bonds between quinones and thiol groups may act as a potential antioxidant by inhibiting disulfide protein cross-link formation.

Properties of fish skin gelatin film incorporated with seaweed extract

Fish skin gelatin films incorporated with 6% oxygenated seaweed ( Turbinaria ornata) extract (based on protein content) with pHs 9 or 10 were prepared and characterized in comparison with the control film (without seaweed extract). Films incorporated with seaweed extract at both pHs exhibited the higher elongation at break (EAB) than the control film ( p < 0.05). However, no differences in tensile strength (TS) and transparency between films without and with seaweed extract were observed ( p > 0.05). Water vapor permeability (WVP), and film solubility decreased as seaweed extract was incorporated, regardless of pH ( p < 0.05). This was associated with the formation of non-disulfide covalent bond in the film matrix, most likely induced by the interaction between oxidized phenols in seaweed extract and gelatin molecules. Coincidentally, the second transition temperature of gelatin film shifted from 170.54 to 178.36 °C when incorporated with 6% seaweed extract at pH 10. Film without seaweed extract had smooth surface while the rougher surface was noticeable in film incorporated with seaweed extract for both pHs. Thus, the addition of seaweed extract had the impact on the property of fish skin gelatin film, most likely due to the enhanced formation of protein cross-links.

Advanced lipid peroxidation end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for the inhibitors

Reactive carbonyl compounds (RCCs) formed during lipid peroxidation and sugar glycoxidation, namely Advanced lipid peroxidation end products (ALEs) and Advanced Glycation end products (AGEs), accumulate with ageing and oxidative stress-related diseases, such as atherosclerosis, diabetes or neurodegenerative diseases. RCCs induce the 'carbonyl stress' characterized by the formation of adducts and cross-links on proteins, which progressively leads to impaired protein function and damages in all tissues, and pathological consequences including cell dysfunction, inflammatory response and apoptosis. The prevention of carbonyl stress involves the use of free radical scavengers and antioxidants that prevent the generation of lipid peroxidation products, but are inefficient on pre-formed RCCs. Conversely, carbonyl scavengers prevent carbonyl stress by inhibiting the formation of protein cross-links. While a large variety of AGE inhibitors has been developed, only few carbonyl scavengers have been tested on ALE-mediated effects. This review summarizes the signalling properties of ALEs and ALE-precursors, their role in the pathogenesis of oxidative stress-associated diseases, and the different agents efficient in neutralizing ALEs effects in vitro and in vivo. The generation of drugs sharing both antioxidant and carbonyl scavenger properties represents a new therapeutic challenge in the treatment of carbonyl stress-associated diseases.