Protein Carbonyl Adducts Research Articles

Role of reactive carbonyls and superoxide radicals in protein damage by cigarette smoke extracts: Comparison of Heat-not-Burn e-cigarettes to conventional cigarettes

Oxidative protein damage involving carbonylation of respiratory tract proteins typically accompanies exposure to tobacco smoke. Such damage can arise via multiple mechanisms, including direct amino acid oxidation by reactive oxygen species or protein adduction by electrophilic aldehydes. This study investigated the relative importance of these pathways during exposure of a model protein to fresh cigarette emission extracts. Briefly, protein carbonyl adducts were estimated in bovine serum albumin following incubation in buffered solutions with whole cigarette emissions extracts prepared from either a single 1R6F research cigarette or a single “Heat-not-Burn” e-cigarette. Although both extracts caused concentration-dependent protein carbonylation, conventional cigarette extracts produced higher adduct yields than e-cigarette extracts. Superoxide radical generation by conventional and e-cigarette emissions was assessed by monitoring nitro blue tetrazolium reduction and was considerably lower in extracts made from “Heat-Not-Burn” e-cigarettes. The superoxide dismutase/catalase mimic EUK-134 strongly suppressed radical production by whole smoke extracts from conventional cigarettes, however, it did not diminish protein carbonyl adduction when incubating smoke extracts with the model protein. In contrast, edaravone, a neuroprotective drug with strong carbonyl-trapping properties, strongly suppressed protein damage without inhibiting superoxide formation. Although these findings require extension to appropriate cell-based and in vivo systems, they suggest reactive aldehydes in tobacco smoke make greater contributions to oxidative protein damage than smoke phase radicals.

NUTRARET: Effect of 2-Year Nutraceutical Supplementation on Redox Status and Visual Function of Patients With Retinitis Pigmentosa: A Randomized, Double-Blind, Placebo-Controlled Trial.

Oxidative stress plays a major role in the pathogenesis of retinitis pigmentosa (RP). The main goal of this study was to evaluate the effect of 2-year nutritional intervention with antioxidant nutraceuticals on the visual function of RP patients. Secondly, we assessed how nutritional intervention affected ocular and systemic redox status. We carried out a randomized, double-blind, placebo-controlled study. Thirty-one patients with RP participated in the study. RP patients randomly received either a mixture of nutraceuticals (NUT) containing folic acid, vitamin B6, vitamin A, zinc, copper, selenium, lutein, and zeaxanthin or placebo daily for 2 years. At baseline and after 2-year of the nutritional supplementation, visual function, dietetic-nutritional evaluations, serum concentration of nutraceuticals, plasma and aqueous humor concentration of several markers of redox status and inflammation were assessed. Retinal function and structure were assessed by multifocal electroretinogram (mfERG), spectral domain-optical coherence tomography (SD-OCT) and automated visual field (VF) tests. Nutritional status was estimated with validated questionnaires. Total antioxidant capacity, extracellular superoxide dismutase (SOD3), catalase (CAT), and glutathione peroxidase (GPx) activities, protein carbonyl adducts (CAR) content, thiobarbituric acid reactive substances (TBARS) formation (as indicator of lipid peroxidation), metabolites of the nitric oxide (NOX) and cytokine (interleukin 6 and tumor necrosis factor alpha) concentrations were assessed by biochemical and immunological techniques in aqueous humor or/and blood. Bayesian approach was performed to determine the probability of an effect. Region of practical equivalence (ROPE) was used. At baseline, Bayesian analysis revealed a high probability of an altered ocular redox status and to a lesser extent systemic redox status in RP patients compared to controls. Twenty-five patients (10 in the treated arm and 15 in the placebo arm) completed the nutritional intervention. After 2 years of supplementation, patients who received NUT presented better retinal responses (mfERG responses) compared to patients who received placebo. Besides, patients who received NUT showed better ocular antioxidant response (SOD3 activity) and lower oxidative damage (CAR) than those who received placebo. This study suggested that long-term NUT supplementation could slow down visual impairment and ameliorate ocular oxidative stress.

Conserved and species-specific molecular denominators in mammalian skeletal muscle aging

Aging is a complex phenomenon involving functional decline in multiple physiological systems. We undertook a comparative analysis of skeletal muscle from four different species, i.e. mice, rats, rhesus monkeys, and humans, at three different representative stages during their lifespan (young, middle, and old) to identify pathways that modulate function and healthspan. Gene expression profiling and computational analysis revealed that pathway complexity increases from mice to humans, and as mammals age, there is predominantly an upregulation of pathways in all species. Two downregulated pathways, the electron transport chain and oxidative phosphorylation, were common among all four species in response to aging. Quantitative PCR, biochemical analysis, mitochondrial DNA measurements, and electron microscopy revealed a conserved age-dependent decrease in mitochondrial content, and a reduction in oxidative phosphorylation complexes in monkeys and humans. Western blot analysis of key proteins in mitochondrial biogenesis discovered that (i) an imbalance toward mitochondrial fusion occurs in aged skeletal muscle and (ii) mitophagy is not overtly affected, presumably leading to the observed accumulation of abnormally large, damaged mitochondria with age. Select transcript expression analysis uncovered that the skeletal inflammatory profile differentially increases with age, but is most pronounced in humans, while increased oxidative stress (as assessed by protein carbonyl adducts and 4-hydroxynonenal) is common among all species. Expression studies also found that there is unique dysregulation of the nutrient sensing pathways among the different species with age. The identification of conserved pathways indicates common molecular mechanisms intrinsic to health and lifespan, whereas the recognition of species-specific pathways emphasizes the importance of human studies for devising optimal therapeutic modalities to slow the aging process.

Protein Carbonylation and Heat Shock Proteins in Human Skeletal Muscle: Relationships to Age and Sarcopenia

Aging is associated with a gradual loss of muscle mass termed sarcopenia, which has significant impact on quality-of-life. Because oxidative stress is proposed to negatively impact upon musculoskeletal aging, we investigated links between human aging and markers of oxidative stress, and relationships to muscle mass and strength in young and old nonsarcopenic and sarcopenic adults. Sixteen young and 16 old males (further subdivided into “old” and “old sarcopenic”) were studied. The abundance of protein carbonyl adducts within skeletal muscle sarcoplasmic, myofibrillar, and mitochondrial protein subfractions from musculus vastus lateralis biopsies were determined using Oxyblot immunoblotting techniques. In addition, concentrations of recognized cytoprotective proteins (eg, heat shock proteins [HSP], αβ-crystallin) were also assayed. Aging was associated with increased mitochondrial (but not myofibrillar or sarcoplasmic) protein carbonyl adducts, independently of (stage-I) sarcopenia. Correlation analyses of all subjects revealed that mitochondrial protein carbonyl abundance negatively correlated with muscle strength ([1-repetition maximum], p = .02, r 2 = −.16), but not muscle mass (p = .13, r 2 = −.08). Abundance of cytoprotective proteins, including various HSPs (HSP 27 and 70), were unaffected by aging/sarcopenia. To conclude, these data reveal that mitochondrial protein carbonylation increases moderately with age, and that this increase may impact upon skeletal muscle function, but is not a hallmark of (stage-I) sarcopenia, per se.

Modulation of Photochemical Damage in Normal and Malignant Cells by Naturally Occurring Compounds†

Certain phytochemicals, such as the stilbene, resveratrol (RES, found in red grapes and berries), and the triterpenoid, ursolic acid (UA, found in waxy berries and herbs such as rosemary and oregano), have antioxidant, anti-inflammatory and antiproliferative effects. Two human-derived cell lines, hTERT-RPE with a nonmalignant phenotype derived from retinal pigment epithelium, and ATCC CRL-11147 derived from a malignant skin melanoma, were used as in vitro models of photooxidative stress produced by exposure to the broadband output of a 150 W Hg vapor arc lamp at an irradiance of 19-26 mW cm(-2). In untreated cells, UV-VIS broadband light exposure produced a loss of proliferative ability, an activation of NF-κB and an increase in protein carbonyl adducts at 24 h postexposure. Pretreatment of the cells with RES or UA at 1-2 μmsignificantly reduced the amount of phosphorylated NF-κB at 24 h postexposure. RES pretreatment reduced the burden of light-induced protein carbonyl adducts by up to 25% in exposed cells. UA treatment markedly increased the sensitivity of melanoma cells to UV radiation, while conferring some photoprotection to RPE cells. These observations indicate that phytochemicals modulate the cellular response to photochemical stress by interacting with specific cell-signaling pathways.

Δ8‐Tetrahydrocannabivarin prevents hepatic ischaemia/reperfusion injury by decreasing oxidative stress and inflammatory responses through cannabinoid CB2 receptors

Activation of cannabinoid CB(2) receptors protects against various forms of ischaemia-reperfusion (I/R) injury. Δ(8) -Tetrahydrocannabivarin (Δ(8) -THCV) is a synthetic analogue of the plant cannabinoid Δ(9) -tetrahydrocannabivarin, which exhibits anti-inflammatory effects in rodents involving activation of CB(2) receptors. Here, we assessed effects of Δ(8) -THCV and its metabolite 11-OH-Δ(8) -THCV on CB(2) receptors and against hepatic I/R injury. Effects in vitro were measured with human CB(2) receptors expressed in CHO cells. Hepatic I/R injury was assessed in mice with 1h ischaemia and 2, 6 or 24h reperfusion in vivo. Displacement of [(3) H]CP55940 by Δ(8) -THCV or 11-OH-Δ(8) -THCV from specific binding sites in CHO cell membranes transfected with human CB(2) receptors (hCB(2) ) yielded K(i) values of 68.4 and 59.95 nM respectively. Δ(8) -THCV or 11-OH-Δ(8) -THCV inhibited forskolin-stimulated cAMP production by hCB(2) CHO cells (EC(50) = 12.95 and 14.3 nM respectively). Δ(8) -THCV, given before induction of I/R, attenuated hepatic injury (measured by serum alanine aminotransferase and aspartate aminotransferase levels), decreased tissue protein carbonyl adducts, 4-hydroxy-2-nonenal, the chemokines CCL3 and CXCL2,TNF-α, intercellular adhesion molecule 1 (CD54) mRNA levels, tissue neutrophil infiltration, caspase 3/7 activity and DNA fragmentation. Protective effects of Δ(8) -THCV against liver damage were still present when the compound was given at the beginning of reperfusion. Pretreatment with a CB(2) receptor antagonist attenuated the protective effects of Δ(8) -THCV, while a CB(1) antagonist tended to enhance it. Δ(8) -THCV activated CB(2) receptors in vitro, and decreased tissue injury and inflammation in vivo, associated with I/R partly via CB(2) receptor activation. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

4-Hydroxynonenal Inhibits SIRT3 via Thiol-Specific Modification

4-Hydroxynonenal (4-HNE) is an endogenous product of lipid peroxidation known to play a role in cellular signaling through protein modification and is a major precursor for protein carbonyl adducts found in alcoholic liver disease (ALD). In the present study, a greater than 2-fold increase in protein carbonylation of sirtuin 3 (SIRT3), a mitochondrial class III histone deacetylase, is reported in liver mitochondrial extracts of ethanol-consuming mice. The consequence of this in vivo carbonylation on SIRT3 deacetylase activity is unknown. Interestingly, mitochondrial protein hyperacetylation was observed in a time-dependent increase in a model of chronic ethanol consumption; however, the underlying mechanisms for this remain unknown. Tandem mass spectrometry was used to identify and characterize the in vitro covalent modification of rSIRT3 by 4-HNE at Cys(280), a critical zinc-binding residue, and the resulting inhibition of rSIRT3 activity via pathophysiologically relevant concentrations of 4-HNE. Computational-based molecular modeling simulations indicate that 4-HNE modification alters the conformation of the zinc-binding domain inducing minor changes within the active site, resulting in the allosteric inhibition of SIRT3 activity. These conformational data are supported by the calculated binding energies derived from molecular docking studies suggesting the substrate peptide of acetyl-CoA synthetase 2 (AceCS2-K(ac)) and display a greater affinity for native SIRT3 as compared with the 4-HNE adducted protein. The results of this study characterize altered mitochondrial protein acetylation in a mouse model of chronic ethanol ingestion and thiol-specific allosteric inhibition of rSIRT3 resulting from 4-HNE adduction.

Protein Carbonyl and the Methionine Sulfoxide Reductase System

The formation and accumulation of protein-carbonyl by reactive oxygen species may serve as a marker of oxidative stress, aging, and age-related diseases. Enzymatic reversal of the protein-carbonyl modification has not yet been detected. However, an enzymatic reversal of protein-methionine sulfoxide modification exists and is mediated by the methionine sulfoxide reductase (Msr) system. Methionine sulfoxide modifications to proteins may precede the formation of protein-carbonyl adducts because of consequent structural changes that increase the vulnerability of amino acid residues to carbonylation. Supportive evidence for this possibility arises from the elevated protein-carbonyl accumulations observed in organisms, such as yeast and mice, lacking the methionine sulfoxide reductase A (MsrA) enzyme. In addition, advanced age or enhanced oxidative-stress conditions foster the accumulations of protein-carbonyls. This review discusses the possible involvement of methionine sulfoxide formation in the occurrence of protein-carbonyl adducts and their relevance to the aging process and neurodegenerative diseases.

Marginal thiamine deficiency increases oxidative markers in the plasma and selected tissues in F344 rats

Abstract A moderate thiamine deficiency that does not affect weight gain or produce the classical signs of thiamine deficiency may have adverse health effects. We assessed the effect of marginally thiamine-deficient diets by measuring the effects of the diets on an enzyme marker for thiamine adequacy and markers for plasma and tissue exposure to potential endogenous carbonyl toxins. Five groups of male F344 rats were fed an AIN76-based diet containing thiamine at 2, 2.9, 5.2, 11, or 18 mg/kg diet. The animals were killed at 70 days by carbon dioxide inhalation, and cardiac blood, liver, colon, and brain samples were obtained. Erythrocytes were analyzed for transketolase activity; plasma for glyoxal, methylglyoxal, hydroimidazolone, nitrotyrosine, and protein carbonyl adducts; and tissue extracts for the 3 protein adducts. Thiamine pyrophosphate–dependent transketolase activity was inversely correlated with dietary thiamine of less than 5.2 mg/kg diet. Plasma methylglyoxal and protein adduct concentrations also increased significantly at dietary thiamine levels or at thiamine level of less than 2.6 mg/kg diet. The brain, liver, and colon showed similar trends, with evident tissue specificity. Diets marginally deficient in thiamine increase the endogenous formation of carbonyl products at thiamine levels only slightly below the requirement levels, 4 mg/kg diet (0.25 mg/kJ diet).

Adenovirus-Mediated Expression of CYP2E1 Produces Liver Toxicity in Mice

Induction of cytochrome P450 2E1 by ethanol is believed to be one of the central pathways by which ethanol generates a state of oxidative stress and causes hepatotoxicity. In order to evaluate the biochemical and toxicological actions of CYP2E1 and its sensitization of hepatotoxin-induced injury, an adenovirus which can mediate overexpression of CYP2E1 was constructed. Injecting this virus into mice through the tail vein elevated CYP2E1 protein and activity twofold in the liver of the mice compared with the mice injected with Ad-LacZ or saline. Transaminase levels were dramatically increased in mice injected with the CYP2E1 adenovirus. Histological evaluation of liver specimens of mice injected with Ad-2E1 showed liver cell injury. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay demonstrated that more cells were stained positively in the liver of the mice infected with Ad-2E1 than in the liver of the mice infected with Ad-LacZ. 3-Nitrotyrosine protein adducts and protein carbonyl adducts were increased in the liver of the mice infected with Ad-2E1 compared with Ad-LacZ. This potentiated toxicity most likely reflects interactions between CYP2E1- and adenovirus-mediated toxicity pathways. These results show that adenovirus-mediated overexpression of CYP2E1 could induce liver toxicity in mice and suggests a mechanism involving oxidative/nitrosative stress.