Glycoxidation of mammalian whole histone generates highly immunogenic aggregates: Sera of SLE patients contain autoantibodies against aggregates.

Non-enzymatic glycation and oxidation of self-proteins, causing formation and accumulation of advanced glycation end products (AGEs), have been reported in an array of pathologies, including systemic lupus erythematosus (SLE). Such modifications may generate neo-epitopes, break immunological tolerance, and induce antibody response. In this study, we have first analysed the structural modifications of whole histone in the presence of deoxyribose followed by oxidation with hydroxyl radicals. Changes in the secondary and tertiary structure of the whole histone were determined by spectroscopic techniques and biochemical assays. Fluorescence spectroscopy and UPLC-MS showed the generation of AGEs such as carboxymethyl lysine and pentosidine, while DLS and TEM indicated the presence of amorphous AGE-aggregates. Moreover, rabbits immunized with these histone-AGEs exhibited enhanced immunogenicity and ELISA and western immunoblot of IgG antibodies from SLE patients' sera showed a significantly higher specificity towards modified histone-AGEs than the native histone.

Anti-glycating and anti-cytotoxic effect of silibinin on albumin at early glycation: A physiochemical study.

During persistent hyperglycaemia, albumin, one of the major blood proteins, can undergo fast glycation. It can be expected that timely inhibition of protein glycation might be add quality years to diabetic patients' life. Therefore, this study was designed to analyse the role of silibinin to reduced or delay amadori adduct formation at early glycation and its beneficial effect to improve the glycated albumin structure and conformation. We also analysed cytotoxic effect of amadori-albumin in the presence of silibinin on murine macrophage cell line RAW cells by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. Formation of early glycated product (furosine) in all samples was confirmed by LCMS. Albumin incubated with glucose only showed presence of furosine like structure. Albumin treated with silibinin in the presence of glucose did not show such furosine like peak. This LCMS result showed the silibinin play a protective role in the formation of early glycated product. HMF contents were also reduced in the presence of silibinin, when albumin was incubated with increasing concentrations of silibinin (100 and 200 µM) in the presence of glucose. ANS binding fluorescence decrease by increasing silibinin concentrations with amadori-albumin. SDS-PAGE was also showed that no significant difference in the band mobility of albumin treated with silibinin as compared to native albumin. The secondary conformational alteration in amadori-albumin due to silibinin were confirmed by FTIR. This spectrum showed slight shift in amide I and Amide II band in albumin co-incubated with glucose and silibinin as compared to albumin incubated with glucose only. We further discussed about cytotoxic effect of amadori albumin and its prevention by silibinin. MTT assay results demonstrated that amadori-albumin showed cytotoxic effect on RAW cells but silibinin showed protective role and increased the cell viability. Moreover, the results showed that silibinin has anti-glycating potential and playing a role to prevent the formation of Amadori-albumin in-vitro. Silibinin possesses strong anti-glycating capacity and can improve albumin structure and function at early stage. It might be useful in delaying the progression of diabetes mellitus and its secondary complications at early stage.

Albumin from sera of rheumatoid arthritis patients share multiple biochemical, biophysical and immunological properties with in vitro generated glyco-nitro-oxidized-albumin.

The purpose of the present study is to explore the effects of endogenous stressors on structure and function of rheumatoid arthritis (RA) patients' albumin. In contrast to glycated-albumin or nitro-oxidized-albumin, high titre antibodies against glyco-nitro-oxidized-albumin were found in the sera of RA patients. Also, compared to the other two modified forms of albumin, glyco-nitro-oxidized-albumin showed highest percent inhibition. Albumin isolated from RA patients' sera displayed hyperchromicity and quenching of tyrosine and tryptophan fluorescence. Fluorescence spectroscopy studies also revealed the presence of dityrosine and advanced glycation end products in RA patient's albumin. RA patients' albumin showed weaker binding with 1-anilinonaphthalene-8-sulfonic acid dye. Secondary structure alterations were demonstrated by circular dichroism and Fourier transform infrared spectroscopy. Biochemical investigations revealed substantial decline in the availability of free side chains of amino acid residues; increased carbonyls and decreased sulfhydryls in RA patients' albumin. The functional impairment in RA patients' albumin was revealed by their low binding with bilirubin and cobalt. Liquid chromatography mass spectrometry analysis revealed the presence of Nε-(carboxymethyl) lysine and 3-nitrotyrosine in RA patients' albumin. The amyloidogenic aggregation of RA patients' albumin was confirmed by Congo red absorption and thioflavin-T fluorescence assays. The morphology of the aggregates was visualized under scanning and transmission electron microscope. From the above findings, we inferred that endogenous stress in RA patients have modified albumin and produce structural/functional abnormalities. Also, the presence of anti-glyco-nitro-oxidized-albumin antibodies along with other clinical features may be used as biomarker for the diagnosis and assessment of treatment responses in RA patients.Communicated by Ramaswamy H. Sarma.

Studies on the synergistic action of methylglyoxal and peroxynitrite on structure and function of human serum albumin.

Albumin, an important serum protein, is continuously exposed to various oxidizing/nitrating and glycating agents. Depending upon the nature/concentration of reactive species present, the protein may be glycated, oxidized/nitroxidized or glyco-nitro-oxidized. Peroxynitrite is a powerful nitroxidant and has been reported to damage a wide array of macromolecules. On the other hand, methylglyoxal is a very strong reactive dicarbonyl and a potent precursor for the formation of advanced glycation end products under pathological conditions. In certain pathological conditions albumin may be modified by peroxynitrite and methylglyoxal simultaneously. There is dearth of literature suggests that structural/conformational and functional alteration in albumin upon glycation and oxidation/nitroxidation, however the alterations produced by glyco-nitro-oxidation has not yet been explored. Therefore, in this study, simultaneous effect of glycation and nitroxidation on the structure and conformation, vis-a-vis function of albumin was explored. Glyco-nitro-oxidized albumin showed decreased free amino acid content together with decreased affinity of albumin towards cobalt. Molecular docking model and molecular dynamic simulations showed close interaction and formation of stable complexes between methylglyoxal, peroxynitrite and albumin. Formation of carboxymethyl lysine and 3-nitrotyrosine in glyco-nitro-oxidized albumin were confirmed by MALDI-TOF MS and UP-LC MS. Aggregate formation in glyco-nitro-oxidized albumin was visualized by transmission electron microscopy. On the basis of these results, it may be speculated that, albumin modified with endogenously generated methylglyoxal and peroxynitrite might be a driving factor in the progression of heightened inflammatory autoimmune responses. The work presents a ground to study the role of glyco-nitro-oxidized albumin in the pathogenesis and progression of various autoimmune diseases including rheumatoid arthritis. Communicated by Ramaswamy H. Sarma.

Therapeutic role of hesperidin in collagen-induced rheumatoid arthritis through antiglycation and antioxidant activities.

Rheumatoid arthritis (RA), a chronic inflammatory disease, and its exact aetiology is not defined clearly. The free radicals produced in large amount in RA are associated with alteration in molecular structure resulting in glycation of proteins. As a result of glycation, advanced glycation end products (AGEs) produced. In this study, collagen type II suspension was injected into Wistar rats to make RA model of rats. Simultaneously, hesperidin 50 mg kg-1 body weight was orally administrated to the rats for 21 days. X-rays of the rat hind paws were analyzed and found to be significantly effective against bone loss after treatment with hesperindin. Nε -(carboxymethyl)lysine (CML) and pentosidine (PTD) concentrations in collagen-induced RA plasma were determined as 565.29 ± 30.15 and 37.23 ± 1.02 ng ml-1 , respectively, while CML and PTD in IgG were 6.63 ± 0.44 ng mg-1 IgG and 425.33 ± 37.26 ng g-1 IgG, respectively. After treatment with hesperidin, the elevated levels of CML in plasma and in IgG were significantly (p < 0.001) lowered to 450.95 ± 15.05 mg ml-1 and 5.23 ± 0.27 ng mg-1 IgG, respectively. Similarly, concentrations of PTD in plasma and IgG of rats treated with hesperidin were 28.46 ± 1.20 ng ml-1 and 359.35 ± 31.11 ng g-1 IgG, respectively. Thus, after treatment with drug, plasma CML and IgG PTD levels were restored as 93% and 16%, respectively, through free radical scavenging activity of hesperidin resulting in alleviation of RA disease by decreasing the AGEs concentrations. Therefore, use of hesperidin may be useful to alleviate severity of RA disease.

Methylglyoxal-induces multiple stable changes in human serum albumin before forming nephrotoxic advanced glycation end-products: Injury demonstration in human embryonic kidney cells.

The minor fraction of methylglyoxal that is not metabolized in healthy humans reacts with macromolecules to form AGEs. In diabetics, the formation of MG is accelerated; its level may be enhanced multifold. The glyoxalase enzymes responsible for the regular and effective clearance of excess methylglyoxal may become defective in diabetes mellitus leading to its retention in cells and plasma. The methylglyoxal-modified-HSA was prepared, characterised by multiple biophysical techniques and biochemical (s) and its damaging effect was examined on embryonic kidney cell line HEK 293. The UV results showed hyperchromicity in MG-modified-HSA while nitroblue tetrazolium and fluorescence data suggested AGEs formation in comparison to control HSA. Upward shift of negative peaks in CD suggested reduction in α-helicity. Accelerated mobility and diffused broad bands observed in native and SDS polyacrylamide gel, respectively suggest neutralization of some of the positive charges on MG-modified-HSA as well as generation of cross-links. As observed by trypan blue assay, MTT, LDH activity assay, acridine orange, propidium iodide, ethidium bromide, 4',6-diamidino-2-phenylindole (DAPI) staining and ROS measurements, the MG-HSA AGEs caused damage to human embryonic kidney cells. The data suggest that MG-HSA AGEs may trigger powerful inflammatory responses at cellular level which might set the stage for nephrotoxicity in diabetics.

Attenuation of hyperglycemia and amadori products by aminoguanidine in alloxan-diabetic rabbits occurs via enhancement in antioxidant defenses and control of stress.

The micro- and macro-complications in diabetes mellitus (DM) mainly arise from the damage induced by Amadori and advanced glycation end products, as well as the released free radicals. The primary goal of DM treatment is to reduce the risk of micro- and macro-complications. In this study, we looked at the efficacy of aminoguanidine (AG) to prevent the production of early glycation products in alloxan-diabetic rabbits. Type1 DM was induced in rabbits by a single intravenous injection of alloxan (90 mg/kg body weight). Another group of rabbits was pre-treated with AG (100 mg/kg body weight) prior to alloxan injection; this was followed by weekly treatment with 100 mg/kg of AG for eight weeks. Glucose, insulin, and early glycation products (HbA1C and fructosamine) were measured in control, diabetic and AG treated diabetic rabbits. The effects of hyperglycemia on superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), reduced glutathione (rGSH), nitric oxide, lipid peroxides, and protein carbonyl were investigated. Alloxan-diabetic rabbits had lower levels of SOD, CAT, Gpx, and rGSH than control rabbits. Nitric oxide levels were considerably greater. AG administration restored the activities of SOD, CAT, Gpx enzymes up to 70-80% and ameliorated the nitric oxide production. HbA1c and fructosamine levels were considerably lower in AG-treated diabetic rabbits. The observed control of hyperglycemia and amadori adducts in alloxan-diabetic rabbits by AG may be attributed to decrease of stress and restoration of antioxidant defenses.

Estimation of parameter of fractional order COVID-19 SIQR epidemic model.

Epidemic model have been broadly used in different forms for studying and forecasting epidemiological processes the spread of dengue, zika virus , HIV, SARS and recently , the 2019-20 corona virus which is an ongoing pandemic of corona virus disease (COVID-19). In the present paper, an inverse problem to find the parameters for the single term (multi term) fractional order system of an outbreak of COVID-19 is considered. In the starting, we propose a numerical method for fractional order corona virus system based on the Gorenflo-Mainardi-Moretti-Paradisi (GMMP) scheme, and then to find the parameters we use GMMP method and the modified hybrid Nelder-Mead Simplex search and particle swarm optimization algorithm. With the new fractional orders and parameters our fractional order corona virus system is capable to providing numerical results that agree well with the real data.

Carbamylation of human serum albumin generates high-molecular weight aggregates: fine characterization by multi-spectroscopic methods and electron microscopy.

Carbamylation is the non-enzymatic reaction between isocyanic acid and macromolecules (mainly proteins) which results in carbamylation-derived products (CDPs) generation, wherein the macromolecules show altered structure and function. In this study, we examined the modifications caused in human serum albumin (HSA) upon interaction with potassium cyanate (KCNO). HSA was incubated with varying concentrations of KCNO for 6 h at 37 °C. The resultant product was characterized by biochemical and biophysical techniques. Among other changes, the carbamylated-HSA showed homocitrulline generation (LC-MS), increase in mass (DLS), and amyloidogenic aggregate formation (Congo red, SEM, TEM). The Gibb's free energy was calculated to be -2.91 to -3.95 kcal mol-1, suggesting that the binding was spontaneous and energetically favourable. The results indicate that in chronic kidney disease patients, elevated levels of isocyanic acid (formed from urea) may modify the albumin structure and lead to its conversion into amyloidogenic aggregates, thus accelerating kidney damage.

Peroxynitrite-Mediated Structural Changes in Histone H2A: Biochemical and Biophysical Analysis.

BACKGROUND: Peroxynitrite, a nitrating and oxidizing agent, is formed by the interaction between nitric oxide and superoxide radicals. H2A histone is a basic nucleoprotein and is one of the major core histones responsible for packaging DNA. It has been shown that they are highly sensitive to oxidizing and nitrating agents. OBJECTIVE: Nitration of tyrosine residues in proteins by peroxynitrite is regarded as a marker of nitrosative damage. The dityrosine bond, an oxidative covalent cross-link between two tyrosines in protein, is increasingly identified as a marker of oxidative stress, aging and neurodegerative diseases. METHODS: Peroxinitrite-mediated nitration and dinitration in H2A histone was assessed by various biophysical techniques. RESULTS: The data presented in this study showed that the dityrosine content was found to be elevated in H2A histone modified with peroxynitrite. The formation of dityrosine showed a decrease in fluorescence intensity, generation of a new peak in FT-IR, increase in hydrodynamic size, and loss of secondary and tertiary structure of H2A resulting in a partially folded structure. CONCLUSION: We report that H2A may undergo conformational and structural changes under nitrosative and oxidative stress from the deleterious effects of peroxynitrite.

Impact of endogenous stress on albumin structure in systemic lupus erythematosus (SLE) patients.

Systemic lupus erythematosus (SLE) is an inflammatory, autoimmune disorder of unknown etiology. The inflammatory stress in SLE patients may modify macromolecules and produce structural/functional abnormalities. The present study is aimed at examining the consequences of stresses on the structure of albumin in SLE patients. Albumin was isolated from the sera of SLE/healthy subjects. Multiple physicochemical techniques were used to elucidate, structure of albumin. Advanced glycation end products in SLE patients' albumin were identified by the AGE specific fluorescence. Quenching of tryptophan, tyrosine fluorescence and surface protein hydrophobicity was observed in SLE patients' albumin. Protein-bound carbonyls were elevated while free thiol, lysine, arginine, and alpha helicity was found to be decreased in SLE albumin. Furthermore, changes in the secondary structure of SLE albumin were observed as shift in the position of amide I/II bands. Functionality of SLE albumin was also compromised as its cobalt-binding ability was substantially declined. Adduction of moieties was detected by dynamic light scattering (DLS) and confirmed by matrix assisted laser desorption/ionization. DLS, thioflavin T and transmission electron microscopy results confirmed aggregates in SLE patients' albumin. This study may be helpful in understanding the role of modified albumin in the cofounding pathologies associated with SLE.

A study on hepatopathic, dyslipidemic and immunogenic properties of fructosylated-HSA-AGE and binding of autoantibodies in sera of obese and overweight patients with fructosylated-HSA-AGE.

Over consumption of fructose may lead to obesity and dyslipidemia and cause fructosylation-induced alterations in the structure and function of proteins. The aim of this study was to investigate the role of fructosylated-HSA-AGE in the pathogenesis of fatty liver (NAFLD and NASH) by biochemical, immunological and histological studies. Immunogenicity of fructosylated-HSA-AGE was probed by inducing antibodies in rabbits. Fructosylated-HSA-AGE was found to be highly immunogenic. Furthermore, fructosylated-HSA-AGE caused mild fibrosis with steatosis and portal inflammation of hepatocytes in experimental animals. Liver function test and dyslipidemic parameters in immunized animals were also found to be raised. Ultrasonography, which should form part of the assessment of chronically raised transaminases, shows fatty infiltration. Interestingly, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, total cholesterol (TC) and triglyceride (TG) profiles confirms USG images of overweight, obese patients. Thus, present study demonstrates that fructosylated-HSA-AGE is hepatotoxic, immunologically active and may cause dyslipidemia.

Physicochemical characterization of carbamylated human serum albumin: an in vitro study.

Carbamylation is an ubiquitous process in which cyanate (OCN-) reacts with the N-terminal amino or ε-amino moiety and generates α-carbamyl amino acids and ε-carbamyl-lysine (homocitrulline). The process leads to irreversible changes in protein charge, structure and function. In this study, we have investigated the effect of carbamyl (generated from potassium cyanate) on human serum albumin (HSA) structure and function. The carbamylated-HSA (c-HSA) showed various modifications when examined by UV, fluorescence, FT-IR and far-UV CD spectroscopies. c-HSA exhibited hypochromicity, loss in α-helical content, changes in the amide I and amide II band, etc. Native-PAGE showed increase in the mobility of c-HSA compared to native-HSA. Aggregate(s) formation in c-HSA was detected by thioflavin T dye. The biochemical investigations carried out on c-HSA suggested increase in carbonyl content and decreased binding of TNBS (trinitrobenzenesulphonic acid) and Sakaguchi reagent. The attachment of the carbamyl moiety to HSA was confirmed from MALDI-TOF results. The functional defects in c-HSA were confirmed from the low binding of bilirubin. Taken together, carbamylation of albumin caused changes in the structural and functional properties of HSA. To the best of our knowledge, this is the first report on detailed biophysical characterization of carbamylated-HSA.

Nitroxidized-Albumin Advanced Glycation End Product and Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease whose major clinical consequence is inflammation of small joints and contiguous structures. Oxidative and nitrosative stress along with increased formation of advanced glycation end products (AGEs) play an important role in the disease process. Generation of reactive species during glycation of proteins further adds to the oxidative and nitrosative stress. Albumin, being the most abundant plasma protein, is frequently targeted by different oxidizing and nitrating agents, including peroxynitrite (OONO-) anion. Albumin is also targeted and modified by dicarbonyl metabolites (glyoxal and methylglyoxal) which are formed in oxidative and non-oxidative processes during the synthesis of AGEs. The endogenously formed OONO- and dicarbonyls may modify plasma albumin including those albumin that have travelled or migrated to synovial cells and caused nitration, oxidation, and glycation. These modifications may produce crosslinks, aggregate in albumin and confer immunogenicity. Simultaneous modification of albumin by OONO- and dicarbonyls may generate nitroxidized-AGE-albumin which may persist in circulation for a longer duration compared to native albumin. Nitroxidized-AGE-albumin level (or serum autoantibodies against nitroxidized- AGE-albumin) along with other pre-clinical features may help predict the likely onset of RA.

A study on correlation between oxidative stress parameters and inflammatory markers in type 2 diabetic patients with kidney dysfunction in north Indian population.

BACKGROUND: Research reports support the statement that oxidative stress and inflammation are well-known risk factors for chronic kidney disease (CKD) in patients with diabetes. This study was designed to ascertain the associated role of oxidative stress parameters and inflammatory markers in diabetes and related CKD among the north Indian population. METHODS: The study was divided into three groups as healthy subjects (group 1), patients with diabetes without complication (group 2), and with CKD (group 3). Serum levels of malondialdehyde (MDA) and nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) content were estimated in all individuals. Inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)-α were determined by enzyme-linked immuno-sorbent assay. RESULTS: MDA, protein carbonyl, and NO were significantly elevated in patients with type 2 diabetes as compared with healthy subjects (P ≤ 0.05). Total thiols content were found to be significantly decreased in patients with diabetes with CKD. The activity of antioxidant enzymes SOD, CAT, and GR showed a significant suppression in patients with type 2 diabetes with or without CKD as compared with healthy subjects. Nevertheless, the levels of proinflammatory cytokines IL-6 and TNF-α were significantly upregulated ( P ≤ 0.05) as compared with healthy subjects. CONCLUSION: Determination of antioxidant defense parameters and inflammatory markers contributes to understand the relationship between oxidative stress and inflammation on the development and prevention of chronic kidney disease in Indian patients with diabetes.

Inhibitory effect of silibinin on Amadori-albumin in diabetes mellitus: A multi-spectroscopic and biochemical approach.

Due to increased understanding of the damaging effects of glycation process, it is highly desirable to manage this process effectively either by prevention or by managing the consequences of glycation preferentially at early stage. The use of potential naturally occurring compounds as anti-glycating agents may provide an effective approach to control the development and progression of diabetic associated complications. In the present study, human serum albumin (albumin) was co-incubated with glucose and different concentrations of silibinin. Silibinin was demonstrated to possess anti-glycation activity. We found that silibinin inhibits glucoseinduced glycation at an early stage. We analyzed the effect of silibinin on albumin structure and its biochemical properties at early stage of glycation through various biophysical and biochemical techniques. Nitro blue tertazolium assay results showed that fructosamine formation was reduced in the presence of silibinin. UV-visible spectra results showed decrease in the absorbance with increasing concentrations of silibinin towards native albumin absorbance. Fluorescence results showed that the intensity was increased with increasing the silibinin concentrations as compared to Amadori-albumin. In addition, Far-UV CD spectra demonstrated some restoration of α-helicity when albumin was incubated with glucose in the presence of silibinin. Moreover, silibinin caused significant reduction in carbonyl contents with concomitant increase in free thiol, lysine and arginine residues. The anti-glycation activity of silibinin was concentration-dependent. From all the observations, we can conclude that silibinin might be acting as an obstacle in the binding of glucose with albumin and thus preventing the glycation induced changes in albumin. Silibinin may be effective in delaying glycation mediated pathologies in diabetic individuals.

Preferential recognition of advanced glycation end products by serum antibodies and low-grade systemic inflammation in diabetes mellitus and its complications.

BACKGROUND: Advanced glycation end products (AGEs) have shown to possess antigenicity. This study analyzes the detrimental effect of non-enzymatic glycation on human serum albumin (HSA) leading to the production of antibodies. METHODS: HSA (20 µM) incubated with d-glucose formed AGEs confirmed by scanning electron microscopy (SEM). DNA-damage was assessed with comet assay. Antibodies against in-vitro formed AGEs was evaluated in the sera of diabetic patients by enzyme-linked immunosorbent assay. Molecular docking was performed to demonstrate affinity of native and glycated-HSA with IgG. Low-grade systemic inflammation was quantified with IL-4, IL-6, TNF-α and NF-Ðºß in serum and mRNA expression. RESULTS: Scanning Electron Microscopy showed the formation of aggregates in glycated-HSA. Comet assay showed DNA damage T2DM with CKD. Serum auto-antibodies in diabetes patients with chronic kidney disease (CKD) showed appreciably high recognition with glycated-HSA compared to native HSA. Molecular docking showed less affinity of glycated-HSA with IgG. Serum IL-4, IL-6, and TNF-α were found significantly higher in T2DM with CKD compared to T2DM and healthy ones. mRNA expression of IL-4, IL-6 and NF-Ðºß are also found significantly higher in T2DM with CKD. CONCLUSION: The non-enzymatic glycation-induced damage to the HSA generate neo-epitopes that possess immunogenic response and low-grade systemic inflammation.

Role of Carbamylated Biomolecules in Human Diseases.

Carbamylation (or carbamoylation) is a non-enzymatic modification of biomolecules mediated by cyanate, a dissociation product of urea. Proteins are more sensitive to carbamylation. Two major sites of carbamylation reaction are: Nα -amino moiety of a protein N-terminus and the Nɛ -amino moiety of proteins' lysine residues. In kidney diseases, urea accumulates and the burden of carbamylation increases. This may lead to alteration in the structure and function of many important proteins relevant in maintenance of homeostasis. Carbamylated proteins namely, carbamylated-haemoglobin and carbamylated-low density lipoprotein (LDL) have been implicated in hypoxia and atherosclerosis, respectively. Furthermore, carbamylation of insulin, oxytocin, and erythropoietin have caused changes in the action of these hormones vis-à-vis the metabolic pathways they control. In this short review, authors have compiled the data on role of carbamylated proteins, enzymes, hormones, LDL, and so on, in human diseases. © 2018 IUBMB Life, 70(4):267-275, 2018.

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.