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Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: Integrated omics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In Saccharomyces cerevisiae, adaptation to hydrogen peroxide (H₂O₂) decreases plasma membrane permeability to H₂O₂, changes its lipid composition and reorganizes ergosterol-rich microdomains by a still unknown mechanism. Here we show, by a quantitative analysis of the H₂O₂-induced adaptation effect on the S. cerevisiae plasma membrane-enriched fraction proteome, using two-dimensional gel electrophoresis, that 44 proteins are differentially expressed. Most of these proteins were regulated at a post-transcriptional level. Fourteen of these proteins contain redox-sensitive cysteine residues and nine proteins are associated with lipid and vesicle traffic. In particular, three proteins found in eisosomes and in the eisosome-associated membrane compartment occupied by Can1p were up-regulated (Pil1p, Rfs1p and Pst2p) during adaptation to H₂O₂. Survival studies after exposure to lethal H₂O₂ doses using yeast strains bearing a gene deletion corresponding to proteins associated to lipid and vesicle traffic demonstrated for the first time that down-regulation of Kes1p, Vps4p and Ynl010wp and up-regulation of Atp1 and Atp2 increases resistance to H₂O₂. Moreover, for the pil1Δ strain, H₂O₂ at low levels produces a hormetic effect by increasing proliferation. In conclusion, these data further confirms the plasma membrane as an active cellular site during adaptation to H₂O₂ and shows that proteins involved in lipid and vesicle traffic are important mediators of H₂O₂ adaptation.

Complementary 2D-PAGE and 'shotgun' LC-MS/MS approaches were combined to identify medium and low-abundant proteins in sera of Cystic Fibrosis (CF) patients (mild or severe pulmonary disease) in comparison with healthy CF-carrier and non-CF carrier individuals aiming to gain deeper insights into the pathogenesis of this multifactorial genetic disease. 78 differentially expressed spots were identified from 2D-PAGE proteome profiling yielding 28 identifications and postulating the existence of post-translation modifications (PTM). The 'shotgun' approach highlighted altered levels of proteins actively involved in CF: abnormal tissue/airway remodeling, protease/antiprotease imbalance, innate immune dysfunction, chronic inflammation, nutritional imbalance and Pseudomonas aeruginosa colonization. Members of the apolipoproteins family (VDBP, ApoA-I, and ApoB) presented gradually lower expression from non-CF to CF-carrier individuals and from those to CF patients, results validated by an independent assay. The multifunctional enzyme NDKB was identified only in the CF group and independently validated by WB. Its functions account for ion sensor in epithelial cells, pancreatic secretion, neutrophil-mediated inflammation and energy production, highlighting its physiological significance in the context of CF. Complementary proteomics-based approaches are reliable tools to reveal pathways and circulating proteins actively involved in a heterogeneous disease such as CF., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Hepatitis delta virus (HDV) infects human hepatocytes already infected with the hepatitis B virus increasing about ten fold the risk of cirrhosis and fulminant hepatitis. The lack of an appropriate cell culture system capable of supporting virus replication has so far impaired the detailed investigation of the HDV biology including the identification of host factors involved in pathogenesis. Here, we made use of a HDV cDNA stably transfected cell line, Huh7-D12, in a proteomic approach to identify the changes in the protein expression profiles in human liver cells that arise as a consequence of HDV replication. Total protein extracts from Huh7-D12 cells and of the corresponding non transfected human liver carcinoma cell line, Huh7, were separated by 2-DE. Differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 23 differentially expressed proteins of which 15 were down regulated and 8 up regulated in Huh7-D12 cells. These proteins were found to be involved in different cellular pathways. The down regulation of the histone H1-binding protein and of triosephosphate isomerase was confirmed by Real time PCR, and the up regulation of the La protein and lamin A/C was validated by western blot.

Hepatitis delta virus (HDV) infection of human hepatocytes infected with the hepatitis B virus (HBV) is associated with increased liver damage and risk of fulminant disease. Although considerable progress has been made towards the elucidation of the mechanisms of HDV replication and pathogenesis, little is still known about the host factors involved in the different steps of the replication cycle. Here, we made use of a proteomic approach to analyse the global alterations in protein expression that arise in human hepatocytes separately transfected with each of the HDV components. Huh7 cells were transiently transfected with plasmids that code for the small delta antigen (S-HDAg), large delta antigen (L-HDAg), genomic RNA (gRNA), and antigenomic RNA (agRNA), respectively. Total protein extracts were separated by 2-DE and differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 32 proteins known to be involved in different pathways namely nucleic acid metabolism, protein metabolism, transport, signal transduction, apoptosis, and cell growth. Moreover, the down regulation of hnRNP D, HSP105, and triosephosphate isomerase was further confirmed by Real time PCR.

Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins. Consistent with the epithelial origin of this tissue, some of the identified proteins are epithelial markers (e.g. cytokeratins, palate lung and nasal epithelium clone protein (PLUNC), and squamous cell carcinoma antigen 1). Comparison of this protein profile with the one similarly obtained for CF nasal cells revealed a set of differentially expressed proteins. These included proteins related to chronic inflammation and some others involved in oxidative stress injury. Alterations were also observed in the levels of cytoskeleton proteins, being probably implicated with cytoskeleton organization changes described to occur in CF-airways. Lower levels were found for some mitochondrial proteins suggesting an altered mitochondrial metabolism in CF. Differential expression was also found for two more enzymes that have not been previously associated to CF. Further studies will clarify the involvement of such proteins in CF pathophysiology and whether they are targets for CF therapy.