Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Quantification of major camel milk proteins by capillary electrophoresis

Proteins from dromedary camel milk (CM) produced in Europe were separated and quantified by capillary electrophoresis (CE). CE analysis showed that camel milk lacks b-lactoglobulin and consists of high concentration of a-lactalbumin (2.01 ± 0.02 mg mL-1), lactoferrin (1.74 ± 0.06 mg mL-1) and serum albumin (0.46 ± 0.01 mg mL-1 ). Among caseins, the concentration of b-casein (12.78 ± 0.92 mg mL-1) was found the highest followed by a-casein (2.89 ± 0.29 mg mL-1) while k-casein represented only minor amount (1.67 ± 0.01 mg mL-1). These results were in agreement with sodium dodecyl sulphatepolyacrylamide gel electrophoresis patterns. Overall, CE offers a quick and reliable method for the determination of major CM proteins, which may be responsible for the many nutritional and health properties of CM.

Astringency reduction in red wine by whey proteins

Whey is a by-product of cheese manufacturing and therefore investigating new applications of whey proteins will contribute towards the valorisation of whey and hence waste reduction. This study shows for the first time a detailed comparison of the effectiveness of gelatin and β-lactoglobulin (β-LG) as fining agents. Gelatin was more reactive than whey proteins to tannic acid as shown by both the astringency method (with ovalbumin as a precipitant) and the tannins determination method (with methylcellulose as a precipitant). The two proteins showed similar selectivity for polyphenols but β-LG did not remove as much catechin. The fining agent was removed completely or to a trace level after centrifugation followed by filtration which minimises its potential allergenicity. In addition, improved understanding of protein–tannin interactions was obtained by fluorescence, size measurement and isothermal titration calorimetry (ITC). Overall this study demonstrates that whey proteins have the potential of reducing astringency in red wine and can find a place in enology.

Interactions between the powdery mildew effector BEC1054 and barley proteins identify candidate host targets

There are over 500 candidate secreted effector proteins (CSEPs) or Blumeria effector candidates (BECs) specific to the barley powdery mildew pathogen Blumeria graminis f.sp. hordei. The CSEP/BEC proteins are expressed and predicted to be secreted by biotrophic feeding structures called haustoria. Eight BECs are required for the formation of functional haustoria. These include the RNase-like effector BEC1054 (synonym CSEP0064). In order to identify host proteins targeted by BEC1054, recombinant BEC1054 was expressed in E. coli, solubilized, and used in pull-down assays from barley protein extracts. Many putative interactors were identified by LC-MS/MS after subtraction of unspecific binders in negative controls. Therefore, a directed yeast-2-hybrid assay, developed to measure the effectiveness of the interactions in yeast, was used to validate putative interactors. We conclude that BEC1054 may target several host proteins, including a glutathione-S-transferase, a malate dehydrogenase, and a pathogen-related-5 protein isoform, indicating a possible role for BEC1054 in compromising well-known key players of defense and response to pathogens. In addition, BEC1054 interacts with an elongation factor 1 gamma. This study already suggests that BEC1054 plays a central role in barley powdery mildew virulence by acting at several levels.

OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.

A glimpse of the ERM proteins

In all eukaryotes, the plasma membrane is critically important as it maintains the architectural integrity of the cell. Proper anchorage and interaction between the plasma membrane and the cytoskeleton is critical for normal cellular processes. The ERM (ezrin-radixin-moesin) proteins are a class of highly homologous proteins involved in linking the plasma membrane to the cortical actin cytoskeleton. This review takes a succinct look at the biology of the ERM proteins including their structure and function. Current reports on their regulation that leads to activation and deactivation was examined before taking a look at the different interacting partners. Finally, emerging roles of each of the ERM family members in cancer was highlighted.

Can milk proteins be a useful tool in the management of cardiometabolic health? An updated review of human intervention trials

The prevalence of cardiometabolic diseases is a significant public health burden worldwide. Emerging evidence supports the inverse association between greater dairy consumption and reduced risk of cardiometabolic diseases. Dairy proteins may have in important role in the favourable impact of dairy on human health such as blood pressure (BP) control, blood lipid and glucose control. The purpose of this review is to update and critically evaluate the evidence on the impacts of casein and whey protein in relation to metabolic function. Evidence from acute clinical studies assessing postprandial responses to milk protein ingestion suggests benefits on vascular function independent of BP, as well as improvement in glycaemic homeostasis. Chronic interventions have been less conclusive, with some showing benefits and others indicating a lack of improvement in vascular function. During chronic consumption BP appears to be lowered and both dyslipidaemia and hyperglacaemia seems to be controlled. Limited number of trials investigated the effects of dairy proteins on oxidative stress and inflammation. The beneficial changes in cardiometabolic homeostasis are likely mediated through improvements in insulin resistance, however to gain more detailed understanding on the underlying mechanism of milk proteins warrants further research. The incorporation of meals enriched with dairy protein in the habitual diet may result in the beneficial effects on cardiometabolic health. Nevertheless, future well-designed, controlled studies are needed to investigate the relative effects of both casein and whey protein on BP, vascular function, glucose homeostasis and inflammation.

Effect of extrusion on the emulsifying properties of soybean proteins and pectin mixtures modelled by response surface methodology

The objective of this study was to apply response surface methodology to estimate the emulsifying capacity and stability of mixtures containing isolated and textured soybean proteins combined with pectin and to evaluate if the extrusion process affects these interfacial properties. A simplex-centroid design was applied to the model emulsifying activity index (EAI), average droplet size (D-[4.3]) and creaming inhibition (Cl%) of the mixtures. All models were significant and able to explain more than 86% of the variation. The high predictive capacity of the models was also confirmed. The mean values for EAI, D-[4.3] and Cl% observed in all assays were 0.173 +/- 0.015 mn, 19.2 +/- 1.0 mu m and 53.3 +/- 2.6%, respectively. No synergism was observed between the three compounds. This result can be attributed to the low soybean protein solubility at pH 6.2 (<35%). Pectin was the most important variable for improving all responses. The emulsifying capacity of the mixture increased 41% after extrusion. Our results showed that pectin could substitute or improve the emulsifying properties of the soybean proteins and that the extrusion brings additional advantage to interfacial properties of this combination. (C) 2008 Elsevier Ltd. All rights reserved.

Caffeic and chlorogenic acids in Ilex paraguariensis extracts are the main inhibitors of AGE generation by methylglyoxal in model proteins

The present study concentrates on the evaluation of the anti-glycation effect of some bioactive substances present in yerba mate (Ilex paraguariensis): 5-caffeoylquinic acid, caffeic acid and a sapogenin (oleanolic acid). Bovine serum albumin and histones were incubated in the presence of methylglyoxal with or without the addition of 5-caffeoylquinic acid, caffeic acid and oleanolic acid. After the incubation period, advanced glycation end product (AGE) fluorescence spectra were performed and protein structural changes were evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. Chlorogenic acid, caffeic acid are the main substances responsible for the anti-glycation effect of mate tea. (C) 2009 Elsevier B.V. All rights reserved.

Effects of abscisic acid, ethylene and sugars on the mobilization of storage proteins and carbohydrates in seeds of the tropical tree Sesbania virgata (Leguminosae)

Endospermic legumes are abundant in tropical forests and their establishment is closely related to the mobilization of cell-wall storage polysaccharides. Endosperm cells also store large numbers of protein bodies that play an important role as a nitrogen reserve in this seed. In this work, a systems approach was adopted to evaluate some of the changes in carbohydrates and hormones during the development of seedlings of the rain forest tree Sesbania virgata during the period of establishment. Seeds imbibed abscisic acid (ABA), glucose and sucrose in an atmosphere of ethylene, and the effects of these compounds on the protein contents, alpha-galactosidase activity and endogenous production of ABA and ethylene by the seeds were observed. The presence of exogenous ABA retarded the degradation of storage protein in the endosperm and decreased alpha-galactosidase activity in the same tissue during galactomannan degradation, suggesting that ABA represses enzyme action. On the other hand, exogenous ethylene increased alpha-galactosidase activity in both the endosperm and testa during galactomannan degradation, suggesting an inducing effect of this hormone on the hydrolytic enzymes. Furthermore, the detection of endogenous ABA and ethylene production during the period of storage mobilization and the changes observed in the production of these endogenous hormones in the presence of glucose and sucrose, suggested a correlation between the signalling pathway of these hormones and the sugars. These findings suggest that ABA, ethylene and sugars play a role in the control of the hydrolytic enzyme activities in seeds of S. virgata, controlling the process of storage degradation. This is thought to ensure a balanced flow of the carbon and nitrogen for seedling development.

Storage proteins and cell wall mobilisation in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

The endosperm of seeds of Sesbania virgata (Cav.) Pers. accumulates galactomannan as a cell wall storage polysaccharide. It is hydrolysed by three enzymes, one of them being alpha-galactosidase. A great amount of protein bodies is found in the cytoplasm of endospermic cells, which are thought to play the major role as a nitrogen reserve in this seed. The present work aimed at understanding how the production of enzymes that degrade storage compounds is controlled. We performed experiments with addition of inhibitors of transcription (actinomycin-d and alpha-amanitin) and translation (cycloheximide) during and after germination. In order to follow the performance of storage mobilisation, we measured fresh mass, protein contents and alpha-galactosidase activity. All the inhibitors tested had little effect on seed germination and seedling development. Actinomycin-d and cycloheximide provoked a slight inhibition of the storage protein degradation and concomitantly lead to an elevation of the alpha-galactosidase activity. Although alpha-amanitin showed some effect on seedling development at latter stages, it presented the former effect and did not change galactomannan degradation performance. Our data suggest that some of the proteases may be synthesised de novo, whereas alpha-galactosidase seems to be present in the endosperm cells probably as an inactive polypeptide in the protein bodies, being probably activated by proteolysis when the latter organelle is disassembled. These evidences suggest the existence of a connection between storage proteins and carbohydrates mobilisation in seeds of S. virgata, which would play a role by assuring a balanced afflux of the carbon and nitrogen to the seedling development.