Protein Network Analysis Identifies Changes in the Level of Proteins Involved in Platelet Degranulation, Proteolysis and Cholesterol Metabolism Pathways in AECOPD Patients.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive pulmonary and systemic inflammation. Acute exacerbations of COPD (AECOPD) are associated with acute inflammation and infection, increase in the rates of morbidity and mortality. Previous proteomic studies have focussed on identifying proteins involved in COPD pathogenesis in samples collected from the lung (e.g. lung tissue biopsies, bronchoalveolar lavage and sputum) but not from blood of patients who experienced AECOPD. In this study, plasma was analysed by two independent quantitative proteomics techniques; isobaric tag for relative and absolute quantitation (iTRAQ) and multiple reaction monitoring (MRM) to identify differential expression of circulating proteins in patients with stable COPD (sCOPD) and AECOPD. Firstly, iTRAQ performed on pooled plasma samples from AECOPD, sCOPD, and healthy non-smoking controls (HC) revealed 15 differentially expressed proteins between the 3 groups. MRM subsequently performed on a separate cohort of AECOPD, sCOPD, and HC patients confirmed 9 proteins to be differentially expressed by AECOPD compared to HC (Afamin, alpha-1-antichymotrypsin, Apolipoprotein E, Beta-2-glycoprotein 1, Complement component C9, Fibronectin, Immunoglobulin lambda like polypeptide 5, Inter-alpha-trypsin inhibitor heavy chain H3, Leucine rich alpha-2-glycoprotein 1). Network analysis demonstrates that most of these proteins are involved in proteolysis regulation, platelet degranulation and cholesterol metabolism. In conclusion, several potential plasma biomarkers for AECOPD were identified in this study. Further validation studies of these proteins may elucidate their roles in the development of AECOPD.

Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT).

This study aimed to compare the depth and reproducibility of total proteome and differentially expressed protein coverage in technical duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex, and TMT 6-plex reagents. The analysis was undertaken because comprehensive comparisons of isobaric mass tag reproducibility have not been widely reported in the literature. The highest number of proteins was identified with 4-plex, followed by 8-plex and then 6-plex reagents. Quantitative analyses revealed that more differentially expressed proteins were identified with 4-plex reagents than 8-plex reagents and 6-plex reagents. Replicate reproducibility was determined to be ≥69% for technical duplicates and ≥57% for technical triplicates. The results indicate that running an 8-plex or 6-plex experiment instead of a 4-plex experiment resulted in 26 or 39% fewer protein identifications, respectively. When 4-plex spectra were searched with three software tools-ProteinPilot, Mascot, and Proteome Discoverer-the highest number of protein identifications were obtained with Mascot. The analysis of negative controls demonstrated the importance of running experiments as replicates. Overall, this study demonstrates the advantages of using iTRAQ 4-plex reagents over iTRAQ 8-plex and TMT 6-plex reagents, provides estimates of technical duplicate and triplicate reproducibility, and emphasizes the value of running replicate samples.

Evidence of altered guinea pig ventricular cardiomyocyte protein expression and growth in response to a 5 min in vitro exposure to H(2)O(2).

Oxidative stress and alterations in cellular calcium homeostasis are associated with the development of cardiac hypertrophy. However, the early cellular mechanisms for the development of hypertrophy are not well understood. Guinea pig ventricular myocytes were exposed to 30 microM H(2)O(2) for 5 min followed by 10 units/mL catalase to degrade the H(2)O(2), and effects on protein expression were examined 48 h later. Transient exposure to H(2)O(2) increased the level of protein synthesis more than 2-fold, assessed as incorporation of [(3)H]leucine (n = 12; p < 0.05). Cell size was increased slightly, but there was no evidence of major cytoskeletal disorganization assessed using fluorescence microscopy. Changes in the expression of individual proteins were assessed using iTRAQ protein labeling followed by mass spectrometry analysis (LC-MALDI-MSMS); 669 proteins were identified, and transient exposure of myocytes to H(2)O(2) altered expression of 35 proteins that were predominantly mitochondrial in origin, including TCA cycle enzymes and oxidative phosphorylation proteins. Consistent with changes in the expression of mitochondrial proteins, transient exposure of myocytes to H(2)O(2) increased the magnitude of the mitochondrial NADH signal 10.5 +/- 2.3% compared to cells exposed to 0 microM H(2)O(2) for 5 min followed by 10 units/mL catalase (n = 8; p < 0.05). In addition, metabolic activity was significantly increased in the myocytes 48 h after transient exposure to H(2)O(2), assessed as formation of formazan from tetrazolium salt. We conclude that a 5 min exposure of ventricular myocytes to 30 microM H(2)O(2) is sufficient to significantly alter protein expression, consistent with the development of hypertrophy in the myocytes. Changes in mitochondrial protein expression and function appear to be early sequelae in the development of hypertrophy.

Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome.

Natural killer (NK) cells and cytotoxic T lymphocytes eliminate virally infected and transformed cells. Target cell killing is mediated by the regulated exocytosis of secretory lysosomes, which deliver perforin and proapoptotic granzymes to the infected or transformed cell. Yet despite the central role that secretory lysosome exocytosis plays in the immune response to viruses and tumors, little is known about the molecular machinery that regulates the docking and fusion of this organelle with the plasma membrane. To identify potential components of this exocytic machinery we used proteomics to define the protein composition of the NK cell secretory lysosome membrane. Secretory lysosomes were isolated from the NK cell line YTS by subcellular fractionation, integral membrane proteins and membrane-associated proteins were enriched using Triton X-114 and separated by SDS-PAGE, and tryptic peptides were identified by LC ESI-MS/MS. In total 221 proteins were identified unambiguously in the secretory lysosome membrane fraction of which 61% were predicted to be either integral membrane proteins or membrane-associated proteins. A significant proportion of the proteins identified play a role in vesicular trafficking, including members of both the Rab GTPase and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and protein families. These proteins include Rab27a and the SNARE vesicle-associated membrane protein-7, both of which were enriched in the secretory lysosome fraction and represent potential components of the machinery that regulates the exocytosis of this organelle in NK cells.

Necrotic death without mitochondrial dysfunction-delayed death of cardiac myocytes following oxidative stress.

Oxidative stress has been implicated in cell death in range of disease states including ischemia/reperfusion injury of the heart and heart failure. Here we have investigated the mechanisms of cell death following chronic exposure of cardiac myocytes to oxidative stress initiated by hydrogen peroxide. This exposure induced a delayed form of cell death with ultrastructural changes typical of necrosis, and that was accompanied by the release of lactate dehydrogenase and increased lipid peroxidation. However, this delayed death was not accompanied by the loss of mitochondrial membrane potential or caspase-3 activation. Furthermore, we could demonstrate that this delayed necrosis was at least partially prevented by pre-treatment with the hypertrophic stimuli endothelin-1 or leukemic inhibitory factor. Our results suggest that this delayed form necrosis may also comprise an ordered series of events involving pathways amenable to therapeutic modulation.

A proteomic analysis of the acute effects of high-intensity exercise on skeletal muscle proteins in fasted rats.

Quantitative proteomics is a technique that allows for large-scale comparison of the levels of individual proteins present in a biological sample. This technique has not previously been applied to examine the response of skeletal muscle proteins to an acute bout of exercise. In the present study, quantitative proteomics was applied to investigate whether the levels of individual skeletal muscle proteins are acutely affected by a short bout of high-intensity exercise. Gastrocnemius muscle was sampled from fasted rats either at rest, immediately following 3 min of high-intensity exercise or after 30 min of recovery. Muscle samples were submitted to two-dimensional gel electrophoresis and 61 of the resulting protein spots were selected for quantitative analysis. It was found that skeletal muscle protein levels were generally not acutely affected by a short bout of high-intensity exercise, with only four of the 61 proteins selected for analysis being significantly altered. These altered proteins were identified using liquid chromatography electrospray ionization-tandem mass spectrometry as creatine kinase, troponin T and a combination of heat shock 20 kDa protein and adenylate kinase 1. In conclusion, quantitative proteomics is sensitive enough to detect acute changes in skeletal muscle protein levels in response to exercise. We have found that the levels of most individual skeletal muscle proteins are not immediately altered in response to a short bout of high-intensity exercise and recovery in fasted rats.

Contrasting actions of prolonged mitogen-activated protein kinase activation on cell survival.

Activation of the ERK mitogen-activated protein kinase pathway has been implicated in pro-survival and cellular protective mechanisms, so that chronic ERK activation may be a useful therapeutic strategy. Here, we further explored the consequences of prolonged ERK activation following expression of constitutively active form of MEK, MEK-EE, in cardiac myocytes. We confirmed that chronic MEK-EE overexpression halved myocyte death following glucose deprivation, but surprisingly this was not associated with preserved intracellular ATP levels. Whilst activities of a number of antioxidant enzymes were not altered upon MEK-EE expression, paradoxically Cu/Zn superoxide dismutase activity was almost halved upon MEK-EE expression. When we then exposed myocytes to the superoxide generator menadione, we observed significantly higher death of MEK-EE expressing myocytes. Pre-incubation with U0126 inhibited menadione-induced death. Our results are the first to show that MEK-ERK signalling can act to increase or decrease cell survival, the outcome depending on the form of stress stimulus encountered.

Proteomic analysis reveals different protein changes during endothelin-1- or leukemic inhibitory factor-induced hypertrophy of cardiomyocytes in vitro.

Proteomic analyses are being increasingly used to identify protein changes accompanying changes in cellular function. An advantage of this approach is that it is largely unbiased by prior assumptions on the importance of each protein in the process under investigation. Here we have evaluated the protein changes that accompany the enlargement, or hypertrophy, of cardiomyocytes in culture. We have taken the additional step of comparing the changes that accompany a concentric hypertrophic phenotype stimulated by endothelin-1 exposure and an eccentric hypertrophic phenotype stimulated by leukemic inhibitory factor exposure. Following separation of the protein extracts by two-dimensional gel electrophoresis and staining with colloidal Coomassie Brilliant Blue, we identified 15 protein spots representing 12 proteins that changed in response to endothelin-1. In comparison, 17 protein spots representing 17 proteins changed in response to leukemic inhibitory factor, and 35 protein spots representing 28 proteins did not change under these conditions. Importantly the well established marker of cardiac pathology, atrial natriuretic factor, was identified as a protein up-regulated by both endothelin-1 and leukemic inhibitory factor (2.4+/-0.8- and 2.2+/-0.3-fold, respectively). However, nine of the observed protein changes occurred for only endothelin-1, whereas 11 of the changes occurred only with leukemic inhibitory factor exposure. These two different stimuli are therefore able to elicit unique changes in the protein expression profile of cardiac myocytes. This is consistent with the differences in morphologies noted as well as the different signaling pathways utilized by these different stimuli.

Hypoxia causes downregulation of protein and RNA synthesis in noncontracting Mammalian cardiomyocytes.

The aim was to identify energy-consuming processes, other than contraction, downregulated during moderate hypoxia ( approximately 5 micromol/L, 0.5% O(2)) and severe hypoxia (<0.5 micromol/L, <0.05% O(2)) in isolated neonatal cardiomyocytes. The metabolic response of cardiomyocytes to moderate and severe hypoxia was assessed by measuring rates of energy consumption and energetic status of cells maintained under these conditions. We found that the rates of energy production were decreased during both forms of hypoxia. Decreased rates of energy production under moderate hypoxia were associated with reduced energy wastage through a downregulation of proton leak in the mitochondria. Cellular protein synthesis and RNA synthesis, major energy-consuming pathways, were downregulated only during severe hypoxia, when oxygen concentrations were low enough to induce energetic stress (quantitatively defined as being any situation in which phosphocreatine concentrations had fallen by > or = 40%). Our results suggest that energetic stress is the signal responsible for this downregulation.