Identification of preferential protein targets for carbonylation in human mature adipocytes treated with native or glycated albumin

Oxidative modifications in proteins can participate in the regulation of cellular functions and are frequently observed in numerous states of diseases. Albumin can undergo increased glycation during diabetes. An accumulation of oxidatively modified proteins in human mature adipocytes incubated with glycated albumin has previously been described. This study herein reports the identification of specifically carbonylated targets following separation of the cell proteins by 2D gels, Western blotting and mass spectrometry analyses. It identified eight oxidatively modified proteins, two of which (ACTB and Annexin A2) appeared as significantly more carbonylated in adipocytes treated with glycated albumin than with native albumin. Intracellular stress, evaluated in SW872 cell line, showed an impairment in the protective antioxidant action exerted by native BSA after the glycation of the protein. Decreased proteasome peptidase activities were found in glycated BSA-treated mature adipocytes. The data suggest an association of oxidative damage with the progression of diabetes disorders at the adipocytes level.     

Diversity of protein carbonylation in allergic airway inflammation

Oxidative stress is involved in asthma. This study assessed the carbonylation of sputum proteins in 23 uncontrolled adult asthmatic patients and 23 healthy controls. Carbonylated proteins (68 kDa and 53 kDa) were elevated in asthmatics when compared to controls and the 68-kDa carbonylated protein was significantly correlated with sputum eosinophilia. The kinetics of protein carbonylation in bronchoalveolar lavage fluid (BALF) were then examined in a mouse ovalbumin-induced allergic inflammation model. It was found that the carbonylation of various BALF proteins did not uniformly occur after challenge. The appearance of the 53-kDa carbonylated protein was limited within 24 h, while carbonylation of 68-kDa protein peaked at 48 h and was associated with BALF eosinophilia. Thus, it was demonstrated that the 68-kDa and 53-kDa proteins, corresponding to albumin and α1-antitrypsin, respectively, were specifically carbonylated in allergic inflammation in humans and in mice and that eosinophils may play a role in mediating carbonylation of albumin.     

Chromatographic analysis of acetohexamide binding to glycated human serum albumin

Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used high-performance affinity chromatography to examine the changes in acetohexamide–HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K_a) for the high affinity interactions was in the range of 1.2–2.0 × 10⁵ M⁻¹ and increased in moving from normal HSA to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K_a for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K_a for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.     

Chemiluminescence analysis of antioxidant capacity for serum albumin isolated from healthy or uremic volunteers

Regular hemodialysis treatment induces an elevation in oxidative stress in patients with end‐stage renal failure, resulting in oxidative damage of the most abundant serum protein, albumin. Oxidation of serum albumin causes depletion of albumin reactive thiols, leading to oxidative modification of serum albumin. The aim of this study was to screen the antioxidant capacity of albumins isolated from uremic patients (HD‐ALB) or healthy volunteers (N‐ALB). From high‐performance liquid chromatography spectra, we observed that one uremic solute binds to HD‐ALB via the formation of disulfide bonds between HD‐ALB and the uremic solute. Furthermore, we found using chemiluminescent analysis that the antioxidant capacities for N‐ALB to scavenge reactive oxygen species including singlet oxygen, hypochlorite and hydrogen peroxide were higher than HD‐ALB. Our results suggest that protein‐bound uremic solute binds to albumin via formation of disulfide bonds, resulting in the depletion of albumin reactive thiols. The depletion of albumin reactive thiols leads to a reduced antioxidant capacity of HD‐ALB, implying postmodification of albumin. This situation may reduce the antioxidant capacity of albumin and increase oxidative stress, resulting in increase in complications related to oxidative damage in uremic patients. Copyright © 2016 John Wiley & Sons, Ltd.     

Assessment of temperature effects on beta-aggregation of native and glycated albumin by FTIR spectroscopy and PAGE: relations between structural changes and antioxidant properties

Structural modifications of bovine serum albumin (BSA) induced by heating, and the involvement of glycation of albumin in such processing were studied by using Fourier transform infrared spectroscopy (FTIR) and polyacrylamide gel electrophoresis (PAGE). For native BSA, heating treatments gave rise to beta structures which were amplified to the detriment of alpha-helix form, and which were associated with increased aggregation. A very high correlation was obtained between FTIR Amide I band evolution and aggregation rate parameters, showing the contribution of beta-form in aggregates formation. We further assessed the effect of glycation on protein sensibility to heating treatments. A reduction of conformational changes and aggregation processes was demonstrated for the glycated form of the protein. The antioxidant properties of albumin were evaluated using two different techniques assessing metal binding and free radical neutralizing capacities of the protein. Associations between structural changes in BSA induced by the thermal treatment and its antioxidant activities were established.     

Specificity of age-related carbonylation of plasma proteins in the mouse and rat.

The purpose of this study was to determine (1) whether oxidative damage to plasma proteins in mice and rats, accrued during aging and manifested as carbonyl modifications, was selective or random, and (2) whether the putative carbonylated proteins could be used as markers of oxidative stress and aging. The total protein carbonyl content of the plasma significantly increased with age in mice but not in rats. Immunostaining of mouse plasma proteins, resolved by SDS-PAGE to localize carbonyls, revealed that only two specific proteins exhibited an age-associated increase in carbonylation. These proteins with molecular weights of 68 and 75 kDa, were identified as albumin and transferrin, respectively. In the rat, albumin and a 167-kDa protein, alpha1-macroglobulin (alpha-1M), showed significant age-dependent accrual of carbonylation. In the plasma of middle age Rhesus monkeys, in addition to albumin, a 54-kDa protein showed carbonylation. However, neither transferrin nor alpha-1M were carbonylated in the plasma of Rhesus monkey. Albumin was the only protein that showed carbonylation in all the three species examined. Results of this study indicate that age-associated increase in protein carbonylation is a selective and not a random phenomenon. However, the set of proteins that become carbonylated differs in different species.     

Multispectroscopic studies of the interaction of folic acid with glycated human serum albumin

Abstract

The interaction between glycated human serum albumin (gHSA) and folic acid (FA) was investigated by various spectroscopic techniques, such as fluorescence, circular dichroism, UV–vis absorption spectroscopy and electrophoretic light scattering technique. These methods characterize the binding properties of an albumin–folic acid system. The binding constants values (K_a) at 300 and 310 K are about 10⁴ M^?1. The standard enthalpy change (ΔH) and the standard entropy change (ΔS) were calculated to be ～?20?kJ mol^?1 and ～16 J mol^?1 K^?1, respectively, which indicate characteristic electrostatic interactions between gHSA and folic acid. The CD studies showed that there are no significant conformational changes in the secondary structure of the protein. Moreover, the zeta potential measurements proved that under physiological conditions the gHSA–folic acid complex shows instability. No significant changes in the secondary structure of the protein and reversible drug binding are the desirable effect from pharmacological point of view.

Communicated by Ramaswamy H. Sarma     

Upregulation of oxidative stress markers in human microvascular endothelial cells by complexes of serum albumin and digestion products of glycated casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95°C for 5 h to give AGE‐casein (AGE‐Cas). Simulated stomach and small intestine digestion of AGE‐Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)‐LMM‐AGE‐Cas complexes. Stimulation of human microvascular endothelial cells with BSA‐LMM‐AGE‐Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin‐3 (AGE‐R3), tumor necrosis factor alpha, and a marker of the mitogen‐activated protein kinase pathway (MAPK‐1), as well as p65NF‐κB activation. Cells treated with LMM digestion products of AGE‐Cas significantly increased AGE‐R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA‐LMM‐AGE‐Cas and LMM‐AGE‐Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and downstream inflammatory pathways. AGE‐R3 may protect against these effects. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:364–372, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20301     

Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model

Several approaches were explored for obtaining high sequence coverage in protein modification studies performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA, 66.5kDa) was used as a model protein for this work. Experimental factors considered in this study included the type of matrix used for MALDI-TOF MS, the protein digestion method, and the use of fractionation for peptide digests prior to MALDI-TOF MS analysis. A mixture of alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid was employed as the final matrix for HSA. When used with a tryptic digest, this gave unique information on only half of the peptides in the primary structure of HSA. However, the combined use of three enzyme digests based on trypsin, endoproteinase Lys-C, and endoproteinase Glu-C increased this sequence coverage to 72.8%. The use of a ZipTip column to fractionate peptides in these digests prior to analysis increased the sequence coverage to 97.4%. These conditions made it possible to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein (e.g., glycation sites). For instance, Lys199 was confirmed as a glycation site on normal HSA, whereas Lys536 and Lys389 were identified as additional modification sites on minimally glycated HSA.     

Serum and salivary levels of albumin as diagnostic tools for oral pre-malignancy and oral malignancy

The role of oxygen free radicals in the initiation, promotion and progression of carcinogenesis and the protective role of antioxidants has been a subject of much speculation. There are few studies that report evaluation of serum albumin and only one study in which salivary albumin was found and only one study that reports of salivary albumin in oral Leukoplakia and Oral Squamous Cell Carcinoma (OSCC). We evaluated serum and salivary albumin levels in normal individuals, patients with oral pre-malignancy and patients with oral malignancy, and we compared serum and salivary albumin levels in patients with oral pre-malignancy and oral malignancy. Our study comprised 45 subjects separated into three groups of 15: normal healthy, oral pre-malignancy and oral malignancy patients. Venous blood was drawn and unstimulated saliva was collected early in the morning. Albumin levels were estimated using the bromocresol green method. Serum albumin levels decreased in oral pre-malignancy and oral malignancy cases compared to healthy individuals. Salivary albumin levels increased in oral pre-malignancy and oral malignancy cases compared to healthy individuals. Our results suggest that albumin may play a role in early diagnosis and prognosis of oral pre-malignant and oral malignant tissues.     

Antioxidant and pro-oxidant actions of resveratrol on human serum albumin in the presence of toxic diabetes metabolites: Glyoxal and methyl-glyoxal

Methylglyoxal (MGO) and glyoxal (GO) are attracting considerable attention because of their role in the onset of diabetes symptoms. Therefore, to comprehend the molecular fundamentals of their pathological actions is of the utmost importance. In this study, the molecular interactions between resveratrol (RES) and human serum albumin (HSA) and the ability of the stilbene to counteract the oxidative damage caused by pathological concentrations of MGO and GO to the human plasma protein, was assessed. The oxidation of Cys34 in HSA as well as the formation of specific protein semialdehydes AAS (α-aminoadipic), GGS (γ-glutamic) and the accumulation of Advanced Glycation End-products (AGEs) was investigated. Resveratrol was found to neutralize both α-dicarbonyls by forming adducts detected by HESI-Orbitrap-MS. This antioxidant action was manifested in a significant reduction of AGEs. However, RES-α-dicarbonyl conjugates oxidized Cys34 and lysine, arginine and/or proline by a nucleophilic attack on SH and ε-NH groups in HSA. The formation of specific semialdehydes in HSA after incubation with GO and MGO at pathological concentrations was reported for the first time in this study, and may be used as early and specific biomarkers of the oxidative stress undergone by diabetic patients. The pro-oxidative role of the RES-α-dicarbonyl conjugates should be further investigated to clarify whether this action leads to positive or harmful clinical consequences. The biological relevance of human protein carbonylation as a redox signaling mechanism and/or as a reflection of oxidative damage and disease should also be studied in future works.     

L-Arginine inhibits in vitro nonenzymatic glycation and advanced glycosylated end product formation of human serum albumin

Summary L-Arginine (Arg) has a structure similar to that of aminoguanidine (AG) and may inhibit glycation and advanced glycosylated end product (AGE) formation. Human serum albumin (HSA) (100mg/ml) was incubated for 2 weeks with glucose (200mM) at 37°C or with glucose and equimolar concentrations of Arg, N--acetyl Arg, or AG with or without 25mM diethylenetriaminepentaacetic acid (DTPA). In the absence of DTPA, electrospray ionization mass spectrometry showed a 70% reduction of covalently bound glucose in the presence of Arg and a 30% reduction with AG. Digestibility by trypsin of HSA incubated with glucose and Arg was similar to that of HSA incubated alone. This suggests less covalent modification of HSA in the presence of Arg as compared with the absence of Arg. When incubations contained DTPA, autoradiography showed less¹⁴C labeling of HSA subunits in the presence of Arg and AG. When the-amino group of Arg was blocked with an acetyl group, labeling was similar to that of HSA incubated with glucose, suggesting involvement of the-amino group in the inhibition. Fluorescence of HSA at ex₃₇₀ and em₄₄₀ was reduced with Arg, but AG was more effective than Arg. These results suggest that Arg, like AG, can inhibit glycation and AGE formation.Presented in part at the FASEB meeting, Atlanta, GA, 1991.     

A one-carbon modification of protein lysine associated with elevated oxidative stress in human substantia nigra

We describe for the first time a naturally occurring lysine modification that is converted to methyllysine by reduction with sodium borohydride. This modification is approximately 1.7 times as abundant in soluble proteins from human substantia nigra pars compacta as in proteins from other brain regions, possibly as a result of elevated oxidative stress in the nigra. Proteins from cultured PC12 cells exposed to oxidative stress conditions also contain elevated levels of this lysine modification. The abundance of the naturally occurring modification is roughly 0.08 nmoles/mg protein in either unstressed brain or PC12 cells. Modification levels remain stable in isolated proteins incubated for 2 h at 37 degrees C in pH 7 buffer. We propose that the endogenous modification is the lysine Schiff base, epsilon-N-methylenelysine, and that lysine modifications may result from a reaction with formaldehyde in vivo. Rat brain contains approximately 60 nmoles/g wet weight of formaldehyde, which probably includes both free and reversibly bound forms. Adding approximately 35 microm HCHO to PC12 cell growth medium introduces methylenelysine modifications in cell proteins and impairs cell viability. The existence of this post-translational modification suggests new mechanisms of oxidative stress that may contribute to tissue degeneration, including loss of nigral dopamine neurons during normal aging and in Parkinson's disease.     

Effects of glycated albumin on mesangial cells: evidence for a role in diabetic nephropathy

Nonenzymatically glycated proteins are preferentially transported across the glomerular filtration barrier, and the glomerular mesangium in diabetes is bathed with serum containing increased concentrations of glycated albumin. We investigated effects of glycated albumin on mesangial cells, which are involved in diabetic nephropathy. [³H]-thymidine incorporation was significantly inhibited when murine mesangial cells were grown in culture media containing human serum that had been nonenzymatically glycated by incubation for 4 days with 28 mM glucose. This inhibition was reversed when monoclonal antibodies that selectively react with Amadori products of glycated albumin were added to the culture media. Purified glycated albumin containing Amadori adducts of the glycation reaction induced significant inhibition of thymidine incorporation and stimulation of Type IV collagen secretion compared with cells cultured in the presence of purified nonglycated albumin. These changes were prevented when monoclonal antibodies specifically reactive with fructosyl-lysine epitopes in glycated albumin were added to the cultures. The antibodies had no effect on growth or collagen production in the presence of nonglycated albumin. The results provide the first evidence directly implicating Amadori adducts in glycated albumin in the pathogenesis of diabetic nephropathy, which is characterized by decreased cellularity in association with expansion of the mesangial matrix.     

Spectroscopic and in silico study of binding mechanism of cynidine‐3‐O‐glucoside with human serum albumin and glycated human serum albumin

The drug–serum albumin interaction plays a dominant role in drug efficacy and disposition. The glycation of serum albumin that occurs during diabetes may affect its drug‐binding properties in vivo. In order to evaluate the interactivity characteristics of cyanidin‐3‐O‐glucoside (C3G) with human serum albumin (HSA) and glycated human serum albumin (gHSA), this study was undertaken using multiple spectroscopic techniques and molecular modeling analysis. Time‐resolved fluorescence and the thermodynamic parameters indicated that the quenching mechanism was static quenching, and hydrogen bonding and Van der Waals force were the main forces. The protein fluorescence could be quenched by C3G, whereas the polarity of the fluorophore was not obviously changed. C3G significantly altered the secondary structure of the proteins. Furthermore, the interaction force that existed in the HSA–C3G system was greater than that in the gHSA–C3G system. Fluorescence excitation emission matrix spectra, red edge excitation shift, Fourier transform infrared spectroscopy and circular dichroism spectra provided further evidence that glycation could inhibit the binding between C3G and proteins. In addition, molecular modeling analysis supported the experimental results. The results provided more details for the application of C3G in the treatment of diabetes.     

Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion

Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrP^C into the infectious misfolded isoform PrP^Sc. Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrP^Sc destabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121–231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders.     

Chromatographic studies of changes in binding of sulfonylurea drugs to human serum albumin due to glycation and fatty acids

This report examines the use of high-performance affinity chromatography as a screening tool for studying the change in binding by sulfonylurea drugs to the protein human serum albumin (HSA) during diabetes. The effects of both the non-enzymatic glycation of HSA and the presence of fatty acids on these interactions were considered using a zonal elution format. It was found that there was a significant increase (i.e., 2.7- to 3.6-fold) in the relative retention of several sulfonylurea drugs (i.e., acetohexamide, tolbutamide, glybenclamide and gliclazide) on columns containing normal versus glycated HSA. The addition of various long chain fatty acids to the mobile phase gave the same trend in retention for the tested drugs on both the HSA and glycated HSA columns, generally leading to lower binding. Most of the fatty acids examined produced similar or moderately different relative shifts in retention; however, palmitic acid was found to produce a much larger change in retention on columns containing glycated HSA versus normal HSA under the conditions used in this study.     

Interaction of ochratoxin A with human serum albumin. Binding sites localized by competitive interactions with the native protein and its recombinant fragments

Competitive interactions of ochratoxin A (OTA) and several other acidic compounds were utilized to gain insight into the localization of binding sites and the nature of binding interactions between anionic species and human serum albumin (HSA). Depolarization of OTA fluorescence in the presence of a competing anion was used to quantify ligand-protein interactions. The results obtained were rationalized in terms of OTA displacement from its major binding site. Based on their ability to displace OTA, two distinct groups of the anionic ligands were revealed. The first group contained structurally diverse compounds that shared a common binding site in subdomain IIA (Sudlow Site I). The second group consisted of three non-steroidal anti-inflammatory drugs, which showed much lower affinity to Site I than the OTA dianion. The major site for these drugs was located in domain III. Fluorescence spectroscopy measurements of OTA, warfarin (WAR) and naproxen (NAP) complexes with recombinant proteins corresponding to the domains of HSA (D1-D3) revealed binding to all domains but with different affinities. The binding constants for OTA and WAR decreased in the series D2z.Gt;D3>D1. In contrast, NAP showed the most favorable interaction with D3 and comparable affinities to the two remaining domains. The OTA binding constant for D2, 7.9 x 10(5) M(-1), was smaller than the largest constant for HSA by a factor of approximately 7. The binding constant for OTA with D3, 1.1 x 10(5) M(-1), was very close to that of the secondary binding site for HSA.     

Evidence of both extra- and intracellular cysteine targets of protein modification for activation of RET kinase

By use of a specifically sulfhydryl group-reactive chemical, 1,4-butanediyl-bismethanethiosulfonate (BMTS), we studied the localization of oxidative stress-responsive target cysteines for activation of a receptor-type protein tyrosine kinase, c-RET. The chemical, which reacted with RET proteins on the cell surface for sulfhydryl-linked aggregation, induced autophosphorylation and activation of RET kinase. When extracellular domain-deleted RET mutant (RET-PTC-1) cells were exposed to BMTS, neither the molecular status nor the activity of the enzyme was affected, suggesting that the target cysteines of BMTS to which cells were exposed for reaction are located in the cysteine-rich region of the extracellular domain of RET kinase. Despite this result, the exposure of a subcellular form of c-RET or RET-PTC-1 kinase isolated by immunoprecipitation to BMTS did induce activation of the enzyme. These results suggest that cysteines in both the extracellular and the intracellular domains of RET can work as target sites of accessible BMTS and possibly other oxidative elements for structural modification and activation of RET kinase.