Computational and physicochemical insight into 4-hydroxy-2-nonenal induced structural and functional perturbations in human low-density lipoprotein.

Lipid peroxidation (LPO) is a biological process that frequently occurs under physiological conditions. Undue oxidative stress increases the level of LPO; which may further contribute to the development of cancer. 4-Hydroxy-2-nonenal (HNE), one of the principal by-products of LPO, is present in high concentrations in oxidatively stressed cells. HNE rapidly reacts with various biological components, including DNA and proteins; however, the extent of protein degradation by lipid electrophiles is not well understood. The influence of HNE on protein structures will likely have a considerable therapeutic value. This research elucidates the potential of HNE, one of the most researched phospholipid peroxidation products, in modifying low-density lipoprotein (LDL). In this study, we tracked the structural alterations in LDL by HNE using various physicochemical techniques. To comprehend the stability, binding mechanism and conformational dynamics of the HNE-LDL complex, computational investigations were carried out. LDL was altered in vitro by HNE, and the secondary and tertiary structural alterations were examined using spectroscopic methods, such as UV-visible, fluorescence, circular dichroism and fourier transform infrared spectroscopy. Carbonyl content, thiobarbituric acid-reactive-substance (TBARS) and nitroblue tetrazolium (NBT) reduction assays were used to examine changes in the oxidation status of LDL. Thioflavin T (ThT), 1-anilinonaphthalene-8-sulfonic (ANS) binding assay and electron microscopy were used to investigate aggregates formation. According to our research, LDL modified by HNE results in changes in structural dynamics, oxidative stress and the formation of LDL aggregates. The current investigation must characterize HNE's interactions with LDL and comprehend how it can change their physiological or pathological functions.Communicated by Ramaswamy H. Sarma.

4-Chloro-1,2-phenylenediamine induced structural perturbation and genotoxic aggregation in human serum albumin.

4-Chloro-1,2-phenylenediamine (4-Cl-OPD) is a halogenated aromatic diamine used as a precursor in permanent hair color production. Despite its well-documented mutagenic and carcinogenic effects in various in vitro and in vivo models, its role in fibrillar aggregate formation and their genotoxic effect in therapeutic proteins has received less attention. The significance of human serum albumin (HSA) arises from its involvement in bio-regulatory and transport processes. HSA misfolding and aggregation are responsible for some of the most frequent neurodegenerative disorders. We used various complementary approaches to track the formation of amyloid fibrils and their genotoxic effect. Molecular dynamics study demonstrated the complex stability. The impact of 4-Cl-OPD on the structural dynamics of HSA was confirmed by Raman spectroscopy, X-ray diffraction, HPLC and SDS-PAGE. Fibrilllar aggregates were investigated using Congo red assay, DLS, and SEM. The genotoxic nature of 4-Cl-OPD was confirmed using plasmid nicking assay and DAPI staining, which revealed DNA damage and cell apoptosis. 4-Cl-OPD provides a model system for studying fibrillar aggregation and their genotoxic potential in the current investigation. Future studies should investigate the inhibition of the aggregation/fibrillation process, which may yield valuable clinical insights.

Biophysical changes in methylglyoxal modified fibrinogen and its role in the immunopathology of type 2 diabetes mellitus.

Methylglyoxal (MG), a highly reactive dicarbonyl metabolite gets generated during glucose oxidation and lipid peroxidation, which contributes to glycation. In type 2 diabetes mellitus (T2DM), non-enzymatic glycosylation of proteins mediated by hyperglycemia results in the pathogenesis of diabetes-associated secondary complications via the generation of AGEs. Under in vitro conditions, MG altered the tertiary structure of fibrinogen. High-performance liquid chromatography (HPLC) and liquid chromatography mass spectroscopy (LCMS) studies confirmed the generation of N-(carboxymethyl) lysine, N-(carboxyethyl) lysine, hydroimidazolone, pentosidine and argpyrimidine in the modified protein. The altered fibrinogen structure upon glycation was further confirmed by confocal microscopy and nuclear magnetic resonance spectra (NMR). MG-Fib was found to be more immunogenic, as compared to its native analogue, in the immunological studies conducted on experimental rabbits. Our results reflect the presence of neo-antigenic determinants on modified fibrinogen. Competitive inhibition enzyme-linked immunosorbent assay suggested the presence of neo-epitopes with marked immunogenicity eliciting specific immune response. Binding studies on purified immunoglobulin G (IgG) confirmed the enhanced and specific immunogenicity of MG-Fib. Studies on interaction of MG-Fib with the circulating auto-antibodies from T2DM patients showed high affinity of serum antibodies toward MG-Fib. This study suggests a potent role of glycoxidatively modified fibrinogen in the generation of auto-immune response in T2DM patients.

Biophysical characterization of structural and conformational changes in methylmethane sulfonate modified DNA leading to the frizzled backbone structure and strand breaks in DNA.

Methyl methanesulfonate (MMS) is a highly toxic DNA-alkylating agent that has a potential to damage the structural integrity of DNA. This work employed multiple biophysical and computational methods to report the MMS mediated structural alterations in the DNA (MMS-DNA). Spectroscopic techniques and gel electrophoresis studies revealed MMS induced exposure of chromophoric groups of DNA; methylation mediated anti→syn conformational change, DNA fragmentation and reduced nucleic acid stability. MMS induced single-stranded regions in the DNA were observed in nuclease S1 assay. FT-IR results indicated MMS mediated loss of the assigned peaks for DNA, partial loss of C-O ribose, loss of deoxyribose region, C-O stretching and bending of the C-OH groups of hexose sugar, a progressive shift in the assigned guanine and adenine peaks, loss of thymine peak, base stacking and presence of C-O-H vibrations of glucose and fructose, indicating direct strand breaks in DNA due to backbone loss. Isothermal titration calorimetry showed MMS-DNA interaction as exothermic with moderate affinity. Dynamic light scattering studies pointed towards methylation followed by the generation of single-stranded regions. Electron microscopy pictured the loss of alignment in parallel base pairs and showed the formation of fibrous aggregates in MMS-DNA. Molecular docking found MMS in close contact with the ribose sugar of DNA backbone having non-bonded interactions. Molecular dynamic simulations confirmed that MMS is capable of interacting with DNA at two levels, one at the level of nitrogenous bases and another at the DNA backbone. The study offers insights into the molecular interaction of MMS and DNA.Communicated by Ramaswamy H. Sarma.

Characterization of Glyoxal Modified LDL: Role in the Generation of Circulating Autoantibodies in Type 2 Diabetes Mellitus and Coronary Artery Disease.

AIMS: This study aims to investigate the role of glyoxal modified LDL in the immunopathology of diabetes and cardiovascular disease. BACKGROUND: Glycoxidation of proteins is widely studied in relation to diabetes and cardiovascular disease. OBJECTIVE: This study probed the glyoxal mediated modifications in LDL, analyzed the immunogenicity of the glycated LDL and ascertained the presence of circulating antibodies against modified LDL in patients with type 2 diabetes mellitus (T2DM), coronary artery disease (CAD) and patients with both (T2DM+CAD). METHODS: Glyoxal mediated modifications in LDL were studied by multiple spectroscopic techniques, high-performance liquid chromatography and electron microscopy. Immunization studies were carried in New Zealand rabbits. The presence of antibodies against glyoxal modified LDL in immunized rabbits and human subjects was analyzed by ELISA. RESULTS: Glyoxal altered the structural integrity of LDL and led to the formation of AGEs. It decreased the alpha-helix content of LDL; increased ß sheet formation, increased carbonyl content and decreased free lysine and arginine content. Modified LDL showed aggregation, generation of of Nε- (Carboxymethyl) lysine and the formation of amorphous type aggregates. It exhibited high antigenicity and generated a specific immune response that shared common antigenic determinants with other glycated proteins. Direct binding data showed the presence of anti-glyoxal modified LDL antibodies in patients with T2DM, CAD and patients with both T2DM and CAD. Further analysis in competitive binding assay revealed specific binding characteristics of auto-antibodies. Sera from patients with T2DM+CAD exhibited the highest binding with glyoxal modified LDL. CONCLUSION: Glyoxal-modified LDL has neo-antigenic determinants that cause the generation of circulating antibodies in diabetes and coronary artery disease. The study might have potential relevance in biomarker development.

Hydroxyl radical induced structural perturbations make insulin highly immunogenic and generate an auto-immune response in type 2 diabetes mellitus.

Reactive oxygen species (ROS) cause oxidative damage to proteins and generate deleterious by-products which induce a breakdown of immune tolerance and produce antibodies against host macromolecules with implication in human diseases. This study characterizes the hydroxyl radical (OH) modifications of insulin, evaluates its cytotoxicity and immunogenicity, and probes its role in type 2 diabetes (T2DM) autoimmunity. The results demonstrate susceptibility of insulin to modifications induced by OH, causing exposure of its chromophoric aromatic amino acid residues, quenching of tyrosine fluorescence intensity, loss of α-helix and gain in ß content. Modification causes re-arrangement of native interactions of the aromatic residues in insulin. It enhanced the carbonyl content in insulin, exposed its hydrophobic patches and generated non-fibrillar, amorphous type of aggregates that are cytotoxic in nature. Native insulin induced low titre antibodies in immunized rabbits, whereas OH modified insulin generated a strong immune response. Competitive ELISA studies showed high specificity of antibodies generated against OH modified insulin towards the modified protein. Cross reaction studies showed the presence of common antigenic determinants on various oxidised proteins. Since T2DM patients show increased ROS production, oxidation of insulin is expected to occur, which might amplify autoimmune reactions against insulin. True to the assumption, direct binding ELISA showed the presence of anti-OH insulin circulating antibodies in T2DM patients which are specific for the oxidized insulin. In conclusion, insulin loses structural integrity to OH, forms cytotoxic amorphous aggregates, turns highly immunogenic and elicits humoral response in T2DM patients.

Withdrawal Notice: Characterization of Glyoxal Modified LDL: Role in the Generation of Circulating Autoantibodies in Type 2 Diabetes Mellitus and Coronary Artery Disease

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Mechanical characterization of native and sugar-modified decellularized kidneys.

Decellularized organs have the potential to be used as scaffolds for tissue engineering organ replacements. The mechanical properties of the extracellular matrix (ECM) following decellularization are critical for structural integrity and for regulation of cell function upon recellularization. Advanced glycation end products (AGEs) accumulate in the ECM with age and their formation is accelerated by several pathological conditions including diabetes. Some AGEs span multiple amino acids to form crosslinks that may alter the mechanical properties of the ECM. The goal of this work was to evaluate how sugar-induced modifications to the ECM affect the mechanical behavior of decellularized kidney. The compressive and tensile properties of the kidney ECM were evaluated using an accelerated model of AGE formation by ribose. Results show that ribose modifications significantly alter the mechanical behavior of decellularized kidney. Increased resistance to deformation corresponds to increased ECM crosslinking, and mechanical changes can be partially mitigated by AGE inhibition. The degree of post-translational modification of the ECM is dependent on the age and health of the organ donor and may play a role in regulating the mechanical properties of decellularized organs.

Glycoxidation of LDL Generates Cytotoxic Adducts and Elicits Humoral Response in Type 2 Diabetes Mellitus.

This study elucidates the immunological implications of methylglyoxal (MGO) modified LDL in diabetes type 2 patients (T2DM). Under in-vitro modifications, MGO altered the tertiary structure of LDL. TNBS and phenanthrenequinone assays confirmed lysine and arginine residues as main targets of MGO in LDL. HPLC and LCMS studies confirmed the generation of Nϵ-(carboxymethyl) lysine in the modified protein. Comet assay showing increased tail length of DNA in lymphocytes inferred the cytotoxicity of MGO-LDL. The easy penetration of MGO-LDL into the nucleus is possibly a consequence of its reduced size, post-modification, as observed from the studies on hydrodynamic radii studies in DLS experiments. MGO-LDL was found to be more immunogenic, as compared to native LDL, in immunological studies conducted on experimental rabbits. Our results reflect the presence of neo-antigenic determinants on modified LDL. Competitive inhibition ELISA suggested the presence of neo-epitopes with marked immunogenicity eliciting specific immune response. Binding studies on purified IgG confirmed the enhanced and specific immunogenicity of MGO-LDL. Studies on interaction of MGO-LDL with the circulating auto-antibodies from T2DM patients showed high affinity of serum-antibodies towards MGO-LDL. This study suggests a potent role of glycoxidatively modified LDL in the generation of auto-immune response in T2DM patients.

Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients.

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α2M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α2M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α2M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in ß-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α2M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α2M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α2M as compared to the native α2M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α2M and makes it immunogenic. The presence of neo-antigenic determinants on modified α2M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease. Communicated by Ramaswamy H. Sarma.

Effect of syringic acid and syringaldehyde on oxidative stress and inflammatory status in peripheral blood mononuclear cells from patients of myocardial infarction.

Oxidative stress and inflammation are considered as therapeutic targets in myocardial injury. The aim of the present study was to investigate the protective effect of syringic acid (SA) and syringaldehyde (SYD) on peripheral blood mononuclear cells (PBMCs) of myocardial infarction (MI) patients. PBMCs from MI patients were cultured in the presence and absence of SA and SYD. The level of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) was estimated. Reactive oxygen species (ROS) formation, oxidation of lipids, proteins, and activity of antioxidant enzymes were also quantified. To further determine biomolecular changes in treated PBMCs, Fourier transform infrared (FTIR) spectroscopic analysis was done. Molecular docking study was also conducted to evaluate the binding interaction of SA and SYD with various target proteins. SA and SYD treated PBMCs of MI patients showed decreased secretion of TNF-α, IL-6, and NO. Moreover, the content of ROS, level of lipid, and protein oxidation showed diminution by treatment with both the compounds. Enhanced antioxidant defense was also observed in treated PBMCs. The FTIR spectra of treated cells revealed safeguarding effect of SA and SYD on biomolecular structure. The molecular docking analysis displayed significant binding affinity of the two compounds towards TNF-α, IL-6, and antioxidant enzymes. Our findings demonstrated potent antioxidant and anti-inflammatory effects of SA and SYD on PBMCs of MI patients. Thus, SA and SYD supplementation might be beneficial in attenuating oxidative stress and inflammation in MI.

Post-translational modifications on glycated plasma fibrinogen: A physicochemical insight.

Methylglyoxal (MGO) is a highly reactive α-oxoaldehyde. It reacts with basic amino acids of the proteins to form advanced glycation end products (AGEs). Fibrinogen is a soluble multi-domain glycoprotein whose major function is to form fibrin clots that prevent blood loss upon vascular injury. In the present study, fibrinogen was incubated with varying concentration of MGO for 7 days followed by its biochemical and biophysical analysis. Glycated plasma fibrinogen (MGO-fibrinogen); exhibited hyperchromicity, a drop in tryptophan and intrinsic fluorescence, augmented AGE-specific fluorescence and melting temperature. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed decrease in mobility of MGO-fibrinogen. Structural perturbations in secondary and tertiary structure were identified by fourier transform-infrared spectroscopy (FT-IR), followed by far and near-UV circular dichroism (CD). Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF-TOF) mass spectrometry studies suggested increase in molecular mass of MGO-fibrinogen. Amyloid like aggregates were confirmed by Thioflavin T (ThT), Congo red assay (CR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ketoamine moieties, carbonyl content (CO), hydroxymethylfurfural (HMF), superoxide and hydroxyl content were markedly elevated, whereas, total antioxidant capacity (TAC) and free thiol content decreased in MGO-fibrinogen as compared to the native protein. These investigations confirmed the structural and functional alterations in MGO-fibrinogen which leads to different physiological conditions like diabetes mellitus, cardiovascular disease etc.

Methylglyoxal modified IgG generates autoimmune response in rheumatoid arthritis.

The detection of autoantibodies generated against modified proteins that stimulate cellular and humoral immune response has developed a lot of interest in the recent years and a search for biomarkers for the early detection of diseases has increased. IgG protein has earned attention for its possible modifications under hyperglycaemic conditions in rheumatoid arthritis, wherein dicarbonyl stress has been reported to alter the structural integrity of the protein. This report suggests that the interaction of the methylglyoxal with the IgG has consequences in the autoimmunopathology of rheumatoid arthritis. Our molecular docking analysis of methylglyoxal and IgG revealed a close interaction between the two molecules. TNBS studies confirmed the interaction by showing a decline in free lysine-arginine content post-MG modifications in IgG. The modified IgG was thermally more stable and showed the generation of glycation adducts N-epsilon-carboxyethyllysine. Rheumatoid arthritis patients showed enhanced carbonyl stress which was expected to induce structural changes in the epitope makeup of IgG. The ELISA studies and gel retardation assay confirmed auto-antibodies against MG modified IgG (MG-IgG) pointing towards the generation of neoepitopes upon IgG after interaction with MG. This study establishes the IgG modification in RA patients under alter carbonyl concentrations.

Biophysical and biochemical studies on glycoxidatively modified human low density lipoprotein.

Methylglyoxal (MGO), a reactive dicarbonyl metabolite is a potent arginine directed glycating agent which has implications for diabetes-related complications. Dicarbonyl metabolites are produced endogenously and in a state of misbalance, they contribute to cell and tissue dysfunction through protein and DNA modifications causing dicarbonyl stress. MGO is detoxified by glyoxalase 1 (GLO1) system in the cytoplasm. Reactive oxygen species (ROS) are known to aggravate the glycation process. Both the processes are closely linked, and their combined activity is often referred to as "glycoxidation" process. Glycoxidation of proteins has several consequences such as type 2 diabetes mellitus (T2DM), aging etc. In this study, we have investigated the glycation of low-density lipoprotein (LDL) using different concentrations of MGO for varied incubation time periods. The structural perturbations induced in LDL were analyzed by UV-Vis, fluorescence, circular dichroism spectroscopy, molecular docking studies, polyacrylamide gel electrophoresis, FTIR, thermal denaturation studies, Thioflavin T assay and isothermal titration calorimetry. The ketoamine moieties, carbonyl content and HMF content were quantitated in native and glycated LDL. Simulation studies were also done to see the effect of MGO on the secondary structure of the protein. We report structural perturbations, increased carbonyl content, ketoamine moieties and HMF content in glycated LDL as compared to native analog (native LDL). We report the structural perturbations in LDL upon modification with MGO which could obstruct its normal physiological functions and hence contribute to disease pathogenesis and associated complications.

Unsaturated aldehyde, 4-hydroxynonenal (HNE) alters the structural integrity of HSA with consequences in the immuno-pathology of rheumatoid arthritis.

Human serum albumin (HSA) - the most abundant plasma protein plays an important role in the transport of endogenous and exogenous molecules in the body. Its modifications have been implicated in a variety of pathological disorders. We have studied the interaction of HNE with HSA at a molecular level by docking experiment and the results suggest a strong interaction between HNE and HSA. Immunological studies revealed that the circulating auto-antibodies in rheumatoid arthritis (RA) patients have a stronger affinity towards HNE-modified HSA. The HSA isolated from RA patients (RA-HSA) exhibited HNE mediated damage in its secondary and tertiary structure when compared to HSA derived from healthy human subjects (NH-HSA). RA patients presented a significant rise in carbonyls and a considerable decline in free thiol content. Preferential binding of experimentally induced anti-HNE-HSA antibodies to RA-HSA over NH-HSA was observed by ELISA. The results suggest HNE induced structural perturbations in HSA with neoepitopes that generate anti-HNE-HSA antibodies in RA. Hence, HNE-HSA may provide lead towards the development of a biomarker for the disease.

Secondary structural alterations in glucoamylase as an influence of protein aggregation.

Glucoamylase (EC 3.2.1.3) from Aspergillus niger possesses 31% α-helix, 36% ß structure and rest aperiodic structure. A transition of glucoamylase structure in the presence of varying concentrations of glyoxal (GO) and trifluoroethanol (TFE) was studied by using multi-methodological approaches. At 20% GO, glucoamylase exists as molten globule state as evident by high tryptophan and ANS fluorescence, retention of secondary structure and loss of native tertiary structure. This state precedes the onset of the aggregation process and maximum is achieved at the highest concentration i.e. at 90% of GO. In parallel study TFE, on increasing concentration up to 25% induces secondary structure transformation leading to accumulation of intermolecular ß sheets, altered tryptophan environment, high ANS and ThT fluorescence resulting in the formation of glucoamylase aggregates. Isothermal titration calorimetric curve is sigmoidal, indicating the weak binding of GO/TFE and glucoamylase. TEM studies showed that glucoamylase exists as globular and amorphous aggregates at 90% glyoxal and 25% TFE respectively. Further, TFE at 70% causes inhibition of enzyme aggregates; the majority of secondary structures observed at this concentration are α helices. Alpha helices being the main key player relocates glucoamylase native environment as evident by CD, FTIR and TEM. Hence induction of ß sheet promotes protein aggregation and α helices inhibits protein aggregation.