Tyrosinase deficiency increases protein carbonyl content in substantia nigra of mice administered retinol palmitate.

Tyrosinase is a key enzyme for the biosynthesis of melanin pigments in peripheral tissues such as skin and retina. Although tyrosinase activity is specifically detected in melanocytes, several studies have shown the expression and enzymatic activity of tyrosinase in the central nervous system, especially in the midbrain substantia nigra. In the present study, we investigated the antioxidative effects of tyrosinase on protein damage in the substantia nigra of mice. C57BL/10JMsHir (B10) and tyrosinase-deficient albino B10.C-Tyrc/Hir (B10-c) mice were intraperitoneally administered retinol palmitate to induce oxidative stress, and the protein carbonyl content, a hallmark of protein oxidative damage, was examined in the substantia nigra. Retinol palmitate administration was found to decrease catalase activity in the substantia nigra of both B10 and B10-c mice, suggesting the induction of oxidative stress due to imbalanced antioxidant systems. In this model, we found that tyrosinase deficiency markedly increases the protein carbonyl content in the substantia nigra. Thus, we concluded that tyrosinase activity prevents protein damage in the substantia nigra of mice that were challenged with oxidative stress. These findings provide novel insight into the physiological role of tyrosinase in the central nervous system.

Hallmarks of oxidative stress in the livers of aged mice with mild glycogen branching enzyme deficiency.

Glycogen branching enzyme (GBE1) introduces branching points in the glycogen molecule during its synthesis. Pathogenic GBE1 gene mutations lead to glycogen storage disease type IV (GSD IV), which is characterized by excessive intracellular accumulation of abnormal, poorly branched glycogen in affected tissues and organs, mostly in the liver. Using heterozygous Gbe1 knock-out mice (Gbe1+/-), we analyzed the effects of moderate GBE1 deficiency on oxidative stress in the liver. The livers of aged Gbe1+/- mice (22 months old) had decreased GBE1 protein levels, which caused a mild decrease in the degree of glycogen branching, but did not affect the tissue glycogen content. GBE1 deficiency was accompanied by increased protein carbonylation and elevated oxidation of the glutathione pool, indicating the existence of oxidative stress. Furthermore, we have observed increased levels of glutathione peroxidase and decreased activity of respiratory complex I in Gbe1+/- livers. Our data indicate that even mild changes in the degree of glycogen branching, which did not lead to excessive glycogen accumulation, may have broader effects on cellular bioenergetics and redox homeostasis. In young animals cellular homeostatic mechanisms are able to counteract those changes, while in aged tissues the changes may lead to increased oxidative stress.

Effect of Exogenous Zinc on MsrB1 Expression and Protein Oxidation in Human Lens Epithelial Cells.

Aging has been related to zinc deficiency, resulting in protein oxidation and age-related decline of methionine sulfoxide reductase (Msr) activity. This study was designed to investigate the levels of methionine sulfoxide reductase B1 (MsrB1) mRNA and oxidized proteins in human lens epithelial (hLE) cells after treatment with exogenous zinc. The role of exogenous zinc in regulation of MsrB1 gene expression and protein oxidation in hLE cells was studied by MTT assay, oxidized protein measurement kit, and real-time PCR. The results showed that hLE cell viability was significantly decreased by MsrB1 gene knockdown or peroxynitrite (ONOO-) treatment, while it was significantly increased after treatment with exogenous zinc (P < 0.05). Protein carbonyl content in hLE cell by MsrB1 gene knockdown or ONOO- treatment was significantly decreased after treatment with ZnSO4 (P < 0.01). And exogenous zinc could increase the level of MsrB1 in hLE cell under normal (P < 0.001) and oxidative stress (P < 0.01) conditions. In conclusion, exogenous zinc could protect hLE cells against MsrB1 gene knockdown or ONOO--induced cell death by upregulation of MsrB1 involved in the elimination of reactive oxygen species (ROS) and oxidized proteins.

Ouabain attenuates oxidative stress and modulates lipid composition in hippocampus of rats in lipopolysaccharide-induced hypocampal neuroinflammation in rats.

Our study aimed to analyze the effect of ouabain (OUA) administration on lipopolysaccharide (LPS)-induced changes in hippocampus of rats. Oxidative parameters were analyzed in Wistar rats after intraperitoneal injection of OUA (1.8 µg/kg), LPS (200 µg/kg), or OUA plus LPS or saline. To reach our goal, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), in addition to levels of reduced glutathione (GSH), protein carbonyl (PCO) and lipid peroxidation (LPO) were evaluated. We also analyzed the membrane lipid profile and some important lipids for the nervous system, such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid and sphingomyelin. The group that received only LPS showed increased oxidative stress, as evidenced by an increase in LPO (about twice), PCO (about three times) levels, and CAT activity (80%). Conversely, administration of LPS decreased GSH levels (55%), and GPx activity (30%), besides a reduction in the amount of PI (60%) and PC (45%). By other side, OUA alone increased the amount of PI (45%), PE (85%), and PC (70%). All harmful effects recorded were attenuated by OUA, suggesting a protective effect against LPS-induced oxidative stress. The relevance of our results extends beyond changes in oxidative parameters induced by LPS, because nanomolar doses of OUA may be useful in neurodegenerative models. Other studies on other cardenolides and substances related issues, as well as the development of new molecules derived from OUA, could also be useful in general oxidative and/or cellular stress, a condition favoring the appearance of neuronal pathologies.

Irreversible plasma and muscle protein oxidation and physical exercise.

The imbalance between the reactive oxygen (ROS) and nitrogen (RNS) species production and their handling by the antioxidant machinery (low molecular weight antioxidant molecules and antioxidant enzymes), also known as oxidative stress, is a condition caused by physiological and pathological processes. Moreover, oxidative stress may be due to an overproduction of free radicals during physical exercise. Excess of radical species leads to the modification of molecules, such as proteins - the most susceptible to oxidative modification - lipids and DNA. With regard to the oxidation of proteins, carbonylation is an oxidative modification that has been widely described. Several studies have detected changes in the total amount of protein carbonyls following different types of physical exercise, but only few of these identified the specific amino acidic residues targets of such oxidation. In this respect, proteomic approaches allow to identify the proteins susceptible to carbonylation and in many cases, it is also possible to identify the specific protein carbonylation sites. This review focuses on the role of protein oxidation, and specifically carbonyl formation, for plasma and skeletal muscle proteins, following different types of physical exercise performed at different intensities. Furthermore, we focused on the proteomic strategies used to identify the specific protein targets of carbonylation. Overall, our analysis suggests that regular physical activity promotes a protection against protein carbonylation, due to the activation of the antioxidant defence or of the turnover of protein carbonyls. However, we can conclude that from the comprehensive bibliography analysed, there is no clearly defined specific physiological role about this post-translational modification of proteins.

Differences in sperm protein abundance and carbonylation level in bull ejaculates of low and high quality.

In breeding and insemination centres, significant variation in bull ejaculate quality is often observed between individuals and also within the same individual. Low-quality semen does not qualify for cryopreservation and is rejected, generating economic loss. The mechanisms underlying the formation of low-quality ejaculates are poorly understood; therefore, the aim of the present study was to investigate the proteomic differences and oxidative modifications (measured as changes in protein carbonylation level) of bull ejaculates of low and high quality. Flow cytometry and computer-assisted sperm analysis were used to assess differences in viability, reactive oxygen species (ROS) level, and sperm motility. To analyse changes in protein abundance, two-dimensional difference gel electrophoresis (2D-DIGE) was performed. Western blotting in conjunction with two-dimensional electrophoresis (2D-oxyblot) was used to quantitate carbonylated sperm proteins. Proteins were identified using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight spectrometry. High quality ejaculates were characterised by higher sperm motility, viability, concentration, and a lower number of ROS-positive cells (ROS+). We found significant differences in the protein profile between high- and low-quality ejaculates, and identified 14 protein spots corresponding to 10 proteins with differences in abundance. The identified sperm proteins were mainly associated with energetic metabolism, capacitation, fertilisation, motility, and cellular detoxification. High-quality ejaculates were characterised by a high abundance of extracellular sperm surface proteins, likely due to more efficient secretion from accessory sex glands and/or epididymis, and a low abundance of intracellular proteins. Our results show that sperm proteins in low-quality ejaculates are characterised by a high carbonylation level. Moreover, we identified, for the first time, 14 protein spots corresponding to 12 proteins with differences in carbonylation level between low- and high-quality ejaculates. The carbonylated proteins were localised mainly in mitochondria or their immediate surroundings. Oxidative damage to proteins in low-quality semen may be associated with phosphorylation/dephosphorylation disturbances, mitochondrial dysfunction, and motility apparatus disorders. Our results contribute to research regarding the mechanism by which low- and high-quality ejaculates are formed and to the identification of sperm proteins that are particularly sensitive to oxidative damage.

Protective potential of glutathione peroxidase-1 gene against cocaine-induced acute hepatotoxic consequences in mice.

Since the cocaine-induced oxidative stress has been established to lead to hepatotoxicity, we examined the role of the glutathione peroxidase (GPx)-1 gene in cocaine-induced hepatotoxicity. Cocaine treatment significantly increased superoxide dismutase activity in as little as 1 hour, with a maximum level at 6 hours in wild-type mice, while significantly decreasing GPx activity and subsequently inducing oxidative damage (i.e., reactive oxygen species, lipid peroxidation and protein carbonylation). These changes were more prominent in the mitochondrial fraction than in the cytosolic fraction. In contrast, genetic overexpression of GPx-1 significantly attenuated cocaine-induced oxidative damage in mice. Cocaine treatment significantly increased alanine aminotransferase and aspartate aminotransferase levels in the serum. Consistently, cocaine significantly enhanced cleaved caspase-3 expression and intramitochondrial Ca2+ , while significantly reducing mitochondrial transmembrane potential. Cocaine treatment potentiated cleavage of protein kinase C δ (PKCδ), mitochondrial translocation of PKCδ, cytosolic release of cytochrome c and activation of caspase-3, followed by hepatopathologic changes. These results were more prominent in GPx-1 knockout than in wild-type mice, and they were less pronounced in overexpressing transgenic than in non-transgenic mice. Combined, our results suggest that the GPx-1 gene possesses protective potential against mitochondrial oxidative burden, mitochondrial dysfunction and hepatic degeneration induced by cocaine and that the protective mechanisms are associated with anti-apoptotic activity via inactivation of PKCδ.

Protective Effect of Kaempferol on the Transgenic Drosophila Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by the accumulation and deposition of ß-amyloid peptides leading to a progressive neuronal damage and cell loss. Besides several hypotheses for explaining the neurodegenerative mechanisms, oxidative stress has been considered to be one of them. Till date, there is no cure for AD, but the pathogenesis of the disease could be delayed by the use of natural antioxidants. In this context, we decided to study the effect of kaempferol against the transgenic Drosophila expressing human amyloid beta-42. METHOD: The AD flies were allowed to feed on the diet having 10, 20, 30 and 40µM of kaempferol for 30 days. After 30 days of exposure, the amyloid beta flies were studied for their climbing ability and Aversive Phototaxis Suppression assay. Amyloid beta flies head homogenate was prepared for estimating the oxidative stress markers, Caspase and acetylcholinesterase activity. RESULTS: The results of the present study reveal that the exposure of AD flies to kaempferol delayed the loss of climbing ability, memory, reduced the oxidative stress and acetylcholinesterase activity. CONCLUSION: Kaempferol could be used as a possible therapeutic agent against the progression of the Alzheimer's disease.

Elevated Nrf-2 responses are insufficient to mitigate protein carbonylation in hepatospecific PTEN deletion mice.

OBJECTIVE: In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease (NASH) is oxidative stress, which leads to the accumulation of highly reactive electrophilic α/ß unsaturated aldehydes. The objective of this study was to determine the impact of NASH on protein carbonylation and antioxidant responses in a murine model. METHODS: Liver-specific phosphatase and tensin homolog (PTEN)-deletion mice (PTENLKO) or control littermates were fed a standard chow diet for 45-55 weeks followed by analysis for liver injury, oxidative stress and inflammation. RESULTS: Histology and Picrosirius red-staining of collagen deposition within the extracellular matrix revealed extensive steatosis and fibrosis in the PTENLKO mice but no steatosis or fibrosis in controls. Increased steatosis and fibrosis corresponded with significant increases in inflammation. PTEN-deficient livers showed significantly increased cell-specific oxidative damage, as detected by 4-hydroxy-2-nonenal (4-HNE) and acrolein staining. Elevated staining correlated with an increase in nuclear DNA repair foci (γH2A.X) and cellular proliferation index (Ki67) within zones 1 and 3, indicating oxidative damage was zonally restricted and was associated with increased DNA damage and cell proliferation. Immunoblots showed that total levels of antioxidant response proteins induced by nuclear factor erythroid-2-like-2 (Nrf2), including GSTµ, GSTπ and CBR1/3, but not HO-1, were elevated in PTENLKO as compared to controls, and IHC showed this response also occurred only in zones 1 and 3. Furthermore, an analysis of autophagy markers revealed significant elevation of p62 and LC3II expression. Mass spectrometric (MS) analysis identified significantly more carbonylated proteins in whole cell extracts prepared from PTENLKO mice (966) as compared to controls (809). Pathway analyses of identified proteins did not uncover specific pathways that were preferentially carbonylated in PTENLKO livers but, did reveal specific strongly increased carbonylation of thioredoxin reductase and of glutathione-S-transferases (GST) M6, O1, and O2. CONCLUSIONS: Results show that disruption of PTEN resulted in steatohepatitis, fibrosis and caused hepatic induction of the Nrf2-dependent antioxidant system at least in part due to elevation of p62. This response was both cell-type and zone specific. However, these responses were insufficient to mitigate the accumulation of products of lipid peroxidation.

Structural changes of fibrinogen as a consequence of cirrhosis.

Cirrhosis is a disease which may develop as a consequence of various conditions. In advanced liver disease, blood coagulation can be seriously affected. Portal hypertension, vascular abnormalities and/or a dysbalance in coagulation factors may result in bleeding disorders or in the development of thrombosis. Fibrinogen is the main protein involved in clot formation and wound healing. The aim of this work was to analyse the glycosylation pattern of the isolated fibrinogen molecules by lectin-based protein microarray, together with the carbonylation pattern of the individual fibrinogen chains, possible changes in the molecular secondary and tertiary structure and reactivity with the insulin-like growth factor-binding protein 1 (IGFBP-1) in patients with cirrhosis. The results pointed to an increase in several carbohydrate moieties: tri/tetra-antennary structures, Gal ß-1,4 GlcNAc, terminal α-2,3 Sia and α-1,3 Man, and a decrease in core α-1,6 Fuc and bi-antennary galactosylated N-glycans with bisecting GlcNAc. Fibrinogen Aα chain was the most susceptible to carbonylation, followed by the Bß chain. Cirrhosis induced additional protein carbonylation, mostly on the α chain. Spectrofluorimetry and CD spectrometry detected reduction in the α-helix content, protein unfolding and/or appearance of modified amino acid residues in cirrhosis. The amount of complexes which fibrinogen forms with IGFBP-1, another factor involved in wound healing was significantly greater in patients with cirrhosis than in healthy individuals. A more detailed knowledge of individual molecules in coagulation process may contribute to deeper understanding of coagulopathies and the results of this study offer additional information on the possible mechanisms involved in impaired coagulation due to cirrhosis.

Impact of carbonylation on glutathione peroxidase-1 activity in human hyperglycemic endothelial cells.

AIMS: High levels of glucose and reactive carbonyl intermediates of its degradation pathway such as methylglyoxal (MG) may contribute to diabetic complications partly via increased generation of reactive oxygen species (ROS). This study focused on glutathione peroxidase-1 (GPx1) expression and the impact of carbonylation as an oxidative protein modification on GPx1 abundance and activity in human umbilical vein endothelial cells (HUVEC) under conditions of mild to moderate oxidative stress. RESULTS: High extracellular glucose and MG enhanced intracellular ROS formation in HUVECs. Protein carbonylation was only transiently augmented pointing to an effective antioxidant defense in these cells. Nitric oxide synthase expression was decreased under hyperglycemic conditions but increased upon exposure to MG, whereas superoxide dismutase expression was not significantly affected. Increased glutathione peroxidase (GPx) activity seemed to compensate for a decrease in GPx1 protein due to enhanced degradation via the proteasome. Mass spectrometry analysis identified Lys-114 as a possible carbonylation target which provides a vestibule for the substrate H2O2 and thus enhances the enzymatic reaction. INNOVATION: Oxidative protein carbonylation has so far been associated with functional inactivation of modified target proteins mainly contributing to aging and age-related diseases. Here, we demonstrate that mild oxidative stress and subsequent carbonylation seem to activate protective cellular redox signaling pathways whereas severe oxidative stress overwhelms the cellular antioxidant defense leading to cell damage. CONCLUSIONS: This study may contribute to a better understanding of redox homeostasis and its role in the development of diabetes and related vascular complications.

Impaired plasminogen binding in patients with venous thromboembolism: Association with protein carbonylation.

INTRODUCTION: Venous thromboembolism (VTE) is associated with hypofibrinolysis. Its mechanisms are poorly understood. We investigated plasminogen-fibrin interaction and its association with fibrinolytic capacity and protein oxidation/carbonylation in VTE patients. MATERIALS AND METHODS: Plasma-purified plasminogen conversion to plasmin and surface plasmon resonance employed for plasminogen-fibrin interactions were individually evaluated in all healthy controls and non-anticoagulated patients following VTE, 10-23months since the event. We also assessed plasma fibrin clot permeability (Ks), clot lysis time (LT), activators and inhibitors of fibrinolysis together with oxidation/carbonylation markers. RESULTS: VTE patients had impaired plasminogen binding to fibrin (apparent Kd, +290%, p=0.002), reduced rate of plasmin generation (-4.7%, p=0.001), and longer LT (+18.6%, p<0.001) compared with controls. Fibrinogen and Ks were similar in both groups. Apparent Kd correlated with LT (r=0.43, p=0.037), tissue plasminogen activator-plasminogen activator inhibitor 1 (tPA-PAI-1) complexes (r=0.63, p=0.012), and active PAI-1 (r=0.49, p=0.03). Compared with controls, VTE patients had higher thiobarbituric acid reactive substances (TBARS), total protein carbonyl content (PC), and lower total antioxidant capacity (all p<0.001), that all were associated with LT (r=0.61, r=0.56, and r=-0.47, respectively, all p<0.05). Impaired plasminogen binding to fibrin reflected by apparent Kd positively correlated with TBARS (r=0.48, p=0.032) and PC (r=0.54, p=0.013) in the whole group. CONCLUSIONS: Plasminogen-fibrin interactions are altered in young and middle-aged VTE patients, without known thrombophilias, except increased factor VIII. The mechanisms underlying these phenomena remain to be established.

Protein carbonyl concentration as a biomarker for development and mortality in sepsis-induced acute kidney injury.

The objective of the present study was to evaluate protein carbonyl concentration as a predictor of AKI development in patients with septic shock and of renal replacement therapy (RRT) and mortality in patients with SAKI. This was a prospective observational study of 175 consecutive patients over the age of 18 years with septic shock upon Intensive Care Unit (ICU) admission. After exclusion of 46 patients (27 due to AKI at ICU admission), a total of 129 patients were enrolled in the study. Demographic information and blood samples were taken within the first 24 h of the patient's admission to determine serum protein carbonyl concentrations. Among the patients who developed SAKI, the development of AKI was evaluated, along with mortality and need for RRT. The mean age of the patients was 63.3 ± 15.7 years, 47% were male and 51.2% developed SAKI during ICU stay. In addition, protein carbonyl concentration was shown to be associated with SAKI. Among 66 patients with SAKI, 77% died during the ICU stay. Protein carbonyl concentration was not associated with RRT in patients with SAKI. However, the ROC curve analysis revealed that higher levels of protein carbonyl were associated with mortality in these patients. In logistic regression models, protein carbonyl level was associated with SAKI development (OR: 1.416; 95% CI: 1.247-1.609; P<0.001) and mortality when adjusted by age, gender, and APACHE II score (OR: 1.357; 95% CI: 1.147-1.605; P<0.001). In conclusion, protein carbonyl concentration is predictive of AKI development and mortality in patients with SAKI, with excellent reliability.

Advanced Oxidation Protein Products and Carbonylated Proteins as Biomarkers of Oxidative Stress in Selected Atherosclerosis-Mediated Diseases.

OBJECTIVES: The main question of this study was to evaluate the intensity of oxidative protein modification shown as advanced oxidation protein products (AOPP) and carbonylated proteins, expressed as protein carbonyl content (C=O) in abdominal aortic aneurysms (AAA), aortoiliac occlusive disease (AIOD), and chronic kidney disease (CKD). DESIGN AND METHODS: The study was carried out in a group of 35 AAA patients and 13 AIOD patients. However, CKD patients were divided into two groups: predialysis (PRE) included 50 patients or hemodialysis (HD) consisted of 34 patients. AOPP and C=O were measured using colorimetric assay kit, while C-reactive protein concentration was measured by high-sensitivity assay (hsCRP). RESULTS: The concentration of AOPP in both AAA and AIOD groups was higher than in PRE and HD groups according to descending order: AAA~AIOD > HD > PRE. The content of C=O was higher in the PRE group in comparison to AIOD and AAA according to the descending order: PRE~HD > AAA~AIOD. CONCLUSIONS: AAA, AIOD, and CKD-related atherosclerosis (PRE and HD) contribute to the changes in the formation of AOPP and C=O. They may promote modification of proteins in a different way, probably due to the various factors that influence oxidative stress here.

Cerebral Oxidative Stress and Microvasculature Defects in TNF-α Expressing Transgenic and Porphyromonas gingivalis-Infected ApoE-/- Mice.

The polymicrobial dysbiotic subgingival biofilm microbes associated with periodontal disease appear to contribute to developing pathologies in distal body sites, including the brain. This study examined oxidative stress, in the form of increased protein carbonylation and oxidative protein damage, in the tumor necrosis factor-α (TNF-α) transgenic mouse that models inflammatory TNF-α excess during bacterial infection; and in the apolipoprotein knockout (ApoE-/-) mouse brains, following Porphyromonas gingivalis gingival monoinfection. Following 2,4-dinitrophenylhydrazine derivatization, carbonyl groups were detected in frontal lobe brain tissue lysates by immunoblotting and immunohistochemical analysis of fixed tissue sections from the frontotemporal lobe and the hippocampus. Immunoblot analysis confirmed the presence of variable carbonyl content and oxidative protein damage in all lysates, with TNF-α transgenic blots exhibiting increased protein carbonyl content, with consistently prominent bands at 25 kDa (p = 0.0001), 43 kDa, and 68 kDa, over wild-type mice. Compared to sham-infected ApoE-/- mouse blots, P. gingivalis-infected brain tissue blots demonstrated the greatest detectable protein carbonyl content overall, with numerous prominent bands at 25 kDa (p = 0.001) and 43 kDa (p = 0.0001) and an exclusive band to this group between 30-43 kDa* (p = 0.0001). In addition, marked immunostaining was detected exclusively in the microvasculature in P. gingivalis-infected hippocampal tissue sections, compared to sham-infected, wild-type, and TNF-α transgenic mice. This study revealed that the hippocampal microvascular structure of P. gingivalis-infected ApoE-/- mice possesses elevated oxidative stress levels, resulting in the associated tight junction proteins being susceptible to increased oxidative/proteolytic degradation, leading to a loss of functional integrity.

Cross-talk between lipid and protein carbonylation in a dynamic cardiomyocyte model of mild nitroxidative stress.

Reactive oxygen and nitrogen species (ROS/RNS) play an important role in the regulation of cardiac function. Increase in ROS/RNS concentration results in lipid and protein oxidation and is often associated with onset and/or progression of many cardiovascular disorders. However, interplay between lipid and protein modifications has not been simultaneously studied in detail so far. Biomolecule carbonylation is one of the most common biomarkers of oxidative stress. Using a dynamic model of nitroxidative stress we demonstrated rapid changes in biomolecule carbonylation in rat cardiomyocytes. Levels of carbonylated species increased as early as 15min upon treatment with the peroxynitrite donor, 3-morpholinosydnonimine (SIN-1), and decreased to values close to control after 16h. Total (lipids+proteins) vs. protein-specific carbonylation showed different dynamics, with a significant increase in protein-bound carbonyls at later time points. Treatment with SIN-1 in combination with inhibitors of proteasomal and autophagy/lysosomal degradation pathways allowed confirmation of a significant role of the proteasome in the degradation of carbonylated proteins, whereas lipid carbonylation increased in the presence of autophagy/lysosomal inhibitors. Electrophilic aldehydes and ketones formed by lipid peroxidation were identified and relatively quantified using LC-MS/MS. Molecular identity of reactive species was used for data-driven analysis of their protein targets. Combination of different enrichment strategies with LC-MS/MS analysis allowed identification of more than 167 unique proteins with 332 sites modified by electrophilic lipid peroxidation products. Gene ontology analysis of modified proteins demonstrated enrichment of several functional categories including proteins involved in cytoskeleton, extracellular matrix, ion channels and their regulation. Using calcium mobilization assays, the effect of nitroxidative stress on the activity of several ion channels was further confirmed.

Determination of Protein Carbonylation and Proteasome Activity in Seeds.

Reactive oxygen species (ROS) have been shown to be toxic but also function as signaling molecules in a process called redox signaling. In seeds, ROS are produced at different developmental stages including dormancy release and germination. Main targets of oxidation events by ROS in cell are lipids, nucleic acids, and proteins. Protein oxidation has various effects on their function, stability, location, and degradation. Carbonylation represents an irreversible and unrepairable modification that can lead to protein degradation through the action of the 20S proteasome. Here, we present techniques which allow the quantification of protein carbonyls in complex protein samples after derivatization by 2,4-dinitrophenylhydrazine (DNPH) and the determination proteasome activity by an activity-based protein profiling (ABPP) using the probe MV151. These techniques, routinely easy to handle, allow the rapid assessment of protein carbonyls and proteasome activity in seeds in various physiological conditions where ROS may act as signaling or toxic elements.

Circadian modulation of proteasome activity and accumulation of oxidized protein in human embryonic kidney HEK 293 cells and primary dermal fibroblasts.

The circadian system orchestrates the timing of physiological processes of an organism living in daily environmental changes. Disruption of circadian rhythmicity has been shown to result in increased oxidative stress and accelerated aging. The circadian regulation of antioxidant defenses suggests that other redox homeostasis elements such as oxidized protein degradation by the proteasome, could also be modulated by the circadian clock. Hence, we have investigated whether proteasome activities and oxidized protein levels would exhibit circadian rhythmicity in synchronized cultured mammalian cells and addressed the mechanisms underlying this process. Using synchronized human embryonic kidney HEK 293 cells and primary dermal fibroblasts, we have shown that the levels of carbonylated protein and proteasome activity vary rhythmically following a 24h period. Such a modulation of proteasome activity is explained, at least in part, by the circadian expression of both Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the proteasome activator PA28αß. HEK 293 cells showed an increased susceptibility to oxidative stress coincident with the circadian-dependent lower activity of the proteasome. Finally, in contrast to young fibroblasts, no circadian modulation of the proteasome activity and carbonylated protein levels was evidenced in senescent fibroblasts. This paper reports a novel role of the circadian system for regulating proteasome function. In addition, the observation that proteasome activity is modulated by the circadian clock opens new avenues for both the cancer and the aging fields, as exemplified by the rhythmic resistance of immortalized cells to oxidative stress and loss of rhythmicity of proteasome activity in senescent fibroblasts.

Oxidative stress and lipotoxicity.

The α,ß polyunsaturated lipid aldehydes are potent lipid electrophiles that covalently modify lipids, proteins, and nucleic acids. Recent work highlights the critical role these lipids play under both physiological and pathological conditions. Protein carbonylation resulting from nucleophilic attack of lysine, histidine, and cysteine residues is a major outcome of oxidative stress and functions as a redox-sensitive signaling mechanism with roles in autophagy, cell proliferation, transcriptional control, and apoptosis. In addition, protein carbonylation is implicated as an initiating factor in mitochondrial dysfunction and endoplasmic reticulum stress, providing a mechanistic connection between oxidative stress and metabolic disease. In this review, we discuss the generation and metabolism of reactive lipid aldehydes, as well as their signaling roles.

Decreasing translation error rate in Escherichia coli increases protein function.

BACKGROUND: Over-expressed native or recombinant proteins are commonly used for industrial and pharmaceutical purposes, as well as for research. Proteins of interest need to be purified in sufficient quantity, quality and specific activity to justify their commercial price and eventual medical use. Proteome quality was previously positively correlated with ribosomal fidelity, but not on a single protein level. Here, we show that decreasing translational error rate increases the activity of single proteins. In order to decrease the amount of enzyme needed for catalysis, we propose an expression system bearing rpsL141 mutation, which confers high ribosomal fidelity. Using alpha-glucosidase (exo-alpha-1,4-glucosidase) and beta-glucanase (beta-D-glucanase) as examples, we show that proteins purified from Escherichia coli bearing rpsL141 mutation have superior activity compared to those purified from wild type E. coli, as well as some commercially available industrial enzymes. RESULTS: Our results indicate that both alpha-glucosidase and beta-glucanase isolated from E. coli bearing rpsL141 mutation have increased activity compared to those isolated from wild type E. coli. Alpha-glucosidase from rpsL141 background has a higher activity than the purchased enzymes, while beta-glucanase from the same background has a higher activity compared to the beta-glucanase purchased from Sigma, but not compared to the one purchased from Megazyme. CONCLUSION: Reduction of the error rate in protein biosynthesis via ribosomal rpsL141 mutation results in superior functionality of single proteins. We conclude that this is a viable system for expressing proteins with higher activity and that it can be easily scaled up and combined with other expression systems to meet the industrial needs.