Cigarette smoke is a contributory factor for the cardiovascular disease, lung diseases and cancers as the dominant illnesses. The proteomic analysis of bovine aortic endothelial cells (BAECs) exposed ...to cigarette smoke has been performed in the broad (Non-linear pH 3–11) and narrow (Linear pH 4–7) range by using two-dimensional gel electrophoresis (2-DE). Out of an average 950 spots observed in 2-DE gels under pH 3–11, the expression level of 25 proteins significantly increased with an increase of cigarette smoke extract (CSE) level, whereas 21 proteins were strongly down-regulated. In narrow (4–7) pH range analysis, 80 proteins showed a significant change in expression level as compared with control. Some of the proteins were found to show similar spot intensity patterns in both the linear (4–7) and nonlinear (3–11) pH ranges. Most of the proteins identified in both pH conditions were found to be involved in apoptosis, inflammation, transcription modulator, signal transduction pathway, ROS production, cell proliferation and extracellular structure formation. In addition, extracellular structural proteins also responded to apoptotic signaling as an indicator. The findings of the present study can be used as an early biomarker to indicate the risks of cigarette smoke related diseases and also in the design of new therapeutic and diagnostic approaches to control these diseases.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Pulmonary embolism (PE) is a common, potentially fatal disease and its diagnosis is challenging because clinical signs and symptoms are nonspecific. In this study, to investigate protein alterations ...of a rat PE model, total serum proteins collected at different time points were separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bioinformatics analysis of 24 differentially expressed proteins showed that 20 had corresponding protein candidates in the database. According to their properties and obvious alterations after PE, changes of serum concentrations of Hp, Fn, DBP, RBP, and TTR were selected to be reidentified by western blot analysis. Semiquantitative RT−PCR showed DBP, RBP, and TTR to be down-regulated at mRNA levels in livers but not in lung tissues. The low serum concentrations of DBP, RBP, and TTR resulted in the up-regulation of 25(OH)D3, vitamin A, and FT4 (ligands of DBP, RBP, and TTR) after acute PE in rat models. The serum levels of Hp and Fn were detected in patients with DVT/PE and controls to explore their diagnostic prospects in acute PE because the mRNA levels of Hp and Fn were found to be up-regulated both in lung tissues and in livers after acute PE. Our data suggested that the concentration of serum Fn in controls was 79.42 ± 31.57 μg/L, whereas that of PE/DVT patients was 554.43 ± 136.18 μg/L (P < 0.001), and that the concentration of serum Hp in controls was 824.37 ± 235.24 mg/L, whereas that of PE/DVT patients was 2063.48 ± 425.38 mg/L (P < 0.001). The experimental PE rat model selected in this study was more similar to the clinical process than the other existing PE animal models, and the findings indicated instant changes of serum proteins within 48 h after acute PE. The exploration of these differentially expressed proteins or their combination with existent markers such as D-dimer may greatly improve the accuracy of the diagnosis of acute PE, but diagnostic tests are still needed to evaluate the sensitivity and specificity of these markers and also the number of false positives and false negatives. Keywords: comparative proteomics • two dimensional gel electrophoresis • mass spectrometry • pulmonary embolism • haptoglobin • ferritin
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IJS, KILJ, NUK, PNG, UL, UM
Bni1p, implicated in cell polarity control and microtubule regulation during yeast budding, is the Saccharomyces cerevisiae homolog of human Formin-homology proteins, such as FMN1, FMN2, FHOD1, ...FHOD3, FHDC1, GRID2IP, FMNL1, FMNL2, FMNL3, DIAPH1, DIAPH2, DIAPH3, DAAM1 and DAAM2. Cdc50p is necessary for subcellular localization of Bni1p and asymmetrical cell division. Lem3p and Ynr048wp are yeast homologs of Cdc50p; however, mammalian homologs of Cdc50p remained to be identified. Here, we identified and characterized CDC50A (TMEM30A), CDC50B (TMEM30B) and CDC50C (TMEM30C) genes by using bioinformatics. C6orf67 and FLJ33850 were representative human CDC50A and CDC50B cDNAs, respectively. Complete coding sequence of CDC50C cDNA was determined by assembling seven exons within AC129803.3 genome sequence. CDC50A, CDC50B and CDC50C genes were mapped to human chromosome 6q14.1, 14q23.1 and 3q12, respectively. Human CDC50A mRNA was expressed in embryonic stem (ES) cells, placenta, brain and chondrosarcoma, while CDC50B mRNA was expressed in pancreatic islet, kidney, prostate as well as in lung carcinoid, parathyroid tumor, bladder tumor, meningioma and pancreatic cancer. Mouse Cdc50a (2010200I23), Cdc50b (9130011B11) and Cdc50c (4933401B01) cDNAs were also identified. Mammalian CDC50 homologs, including human CDC50A (361 aa), CDC50B (351 aa), CDC50C (341 aa), mouse Cdc50a (364 aa), Cdc50b (353 aa) and Cdc50c (342 aa), were two-transmembrane-spanning proteins with one extracellular loop. Membrane topology and extracellular loop containing three Cys residues and one Asn-linked glycosylation site were evolutionarily conserved among mammalian CDC50 homologs and yeast Cdc50p homologs. Mammalian CDC50 homologs were predicted components of phospholipid-translocators just like yeast Cdc50p and Lem3p.
Hyperinsulinemia is a risk factor in atherosclerosis formation that it stimulated vascular smooth muscle cells (VSMCs) proliferation and migration. To understand the underlying molecular mechanism ...involved in the processes of cellular response to insulin, VSMCs from Wistar-Kyoto rat (WKY) and spontaneous hypertensive rat (SHR) were isolated and cultured, and its proteome was comparatively analyzed with normal control by two-dimensional gel electrophoresis (2-DE). Results showed that the proliferation of VSMCs from SHR be more sensitive to insulin stimulation than that VSMCs from WKY. The detectable spots ranged from 537 to 608 on the gels in VSMCs of SHR, and 413 ± 31 spots in VSMCs of WKY. The different expressed protein spots in VSMCs of SHR were then isolated and measured by matrix-assisted desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). A total of 18 spots showed a sharp clear spectrum, and 13 spots matched with the known proteins from database. These proteins were mainly involved in cytoskeleton, glycometabolism, and post-translational processes. Among these proteins, OPN and matrix gla protein were up-regulated expression proteins, while α-SM actin was down-regulated. Furthermore, these preliminarily identified proteins confirmed by RT-PCR and western blotting analysis were coincident with the changes in 2-DE check. In addition, the cytoskeleton changes and migration rate of VSMCs from SHR treated by insulin increased significantly. The results showed that insulin plays a crucial role in activating proliferation and migration of VSMCs, by regulating the phenotype switch of VSMCs.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The brain and reproductive organ expressed (BRE) gene encodes a highly conserved stress‐modulating protein. To gain further insight into the function of this gene, we used comparative proteomics to ...investigate the protein profiles of C2C12 and D122 cells resulting from small interfering RNA (siRNA)‐mediated silencing as well as overexpression of BRE. Silencing of BRE in C2C12 cells, using siRNA, resulted in up‐regulated Akt‐3 and carbonic anhydrase III expression, while the 26S proteasome regulatory subunit S14 and prohibitin were down‐regulated. Prohibitin is a potential tumour suppressor gene, which can directly interact with p53. We found that cell proliferation was significantly increased after knockdown of BRE, concomitant with reduced p53 and prohibitin expression. In contrast, we observed decreased proliferation and up‐regulation of p53 and prohibitin when BRE was overexpressed in the D122 cell line. In total, five proteins were found to be up‐regulated after BRE over‐expression. The majority of these proteins can target or crosstalk with NF‐κB, which plays a central role in regulating cell proliferation, differentiation and survival. Our results establish a crucial role for BRE in the regulation of key proteins of the cellular stress‐response machinery and provide an explanation for the multifunctional nature of BRE.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Sonic hedgehog (SHH), Indian hedgehog (IHH), and Desert hedgehog (DHH) are key molecules for the integrome network in oncology and regenerative medicine. Soluble Hedgehog ligands bind to Patched ...receptor to activate Smoothened seven-transmembrane receptor with Frizzled domain. KIF27 and KIF7 are human homologs of Drosophila Costal-2 (Cos2), associating with Smoothened, GLI homolog, Fused, and microtubule. Smoothened activation leads to GLI1, GLI2, or GLI3-dependent transcription of Hedgehog target genes. Here, comparative proteomics analyses and comparative genomics analyses on SHH orthologs were performed by using bioinformatics. Human SHH representative transcript was assembled by using BX461534 EST, NM_000193.2 RefSeq, AA503654 EST, and AC078834.5 genome sequence. Human SHH mRNA was expressed in fetal brain, infant brain, and also in colorectal cancer. Chimpanzee SHH gene, consisting of three exons, was located within AC147335.2 genome sequence. Human SHH and chimpanzee SHH (462 aa) showed E284G and T416P amino-acid substitutions. Vertebrate SHH orthologs shared the common domain architecture, consisting of N-terminal signal peptide, Hedgehog signaling domain, Hint domain, and C-terminal HPLGMxxxxS motif. Evolutionarily conserved SHH promoter region (nucleotide position 104429-104083 of human genome sequence AC078834.5) was identified. Double bHLH binding sites, CCAAT box, and TATA box were conserved among human SHH promoter, chimpanzee SHH promoter, rat Shh promoter, and mouse Shh promoter.
Cell−cell interactions play a crucial role during embryogenesis and are enhanced during cell aggregation. P19 mouse embryonic carcinoma cells can differentiate into neural cells by the addition of ...retinoic acid (RA) or by overexpression of the Wnt1 gene, with both processes dependent on cell aggregation. To identify molecules involved in the cell aggregation process, two-dimensional gel electrophoresis (2DE) was used to establish the cell aggregation-associated protein profiles. MALDI-TOF/TOF was used to identify 71 protein spots with differential expression patterns. Among these spots, 54 were differentially expressed in both P19 and Wnt1-overexpressing P19 (Wnt1/P19) cell aggregates, with 42 proteins up-regulated and 12 proteins down-regulated. The other 17 spots were differentially expressed only in Wnt1/P19 cells. The expression patterns of 5 cell aggregation-associated proteins, N-myc downstream-regulated gene 1 (NDRG1), 14-3-3 epsilon, 14-3-3 gamma, acid calponin and cell division control protein 2 homologue (Cdc2), were confirmed by immunoblot and RT-PCR. To further investigate the relationship between cell aggregation and neural differentiation, NDRG1 expression was inhibited by RNA interference during P19 cell aggregation. Silencing of NDRG1 reduced the size of cell aggregates and the expression of N-cadherin, and it also impaired the RA-induced P19 cell neural differentiation. In conclusion, this study provides new clues for the possible mechanism underlying cell aggregation during pluripotent stem cell neural differentiation.
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IJS, KILJ, NUK, PNG, UL, UM
Two-dimensional electrophoresis (2-DE) and shotgun peptide sequencing are the two major technologies to compare the expression profile of proteins, which is also referred to as comparative proteomics ...or quantitative proteomics. Although the methodologies, such as difference gel electrophoresis for 2-DE and isotope-coded affinity tags for shotgun peptide sequencing, have made rapid progress, these two approaches have their own strengths and weaknesses. Therefore, the combination of the two methodologies is beneficial for the purpose of better comparative proteomics, especially in comprehensive coverage of the proteome and protein information such as post-translational modifications.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Agarose based immobilized metal affinity chromatography (IMAC) columns loaded with copper (II) were evaluated for the selection of histidine-containing peptides in comparative proteomics. Recovery, ...binding specificity, and reproducibility were investigated with model proteins. Cu(II)-IMAC was found to be highly selective for histidine containing peptides; moreover, a low degree of nonspecific selection was observed. Acylation of the amino-terminus of peptides with either succinic anhydride, N-acetoxysuccinamide, or 3-(2,5)-dioxopyrrolidin-1-yloxycarbonyl)-propyl-trimethylammonium (quaternary amine) reduced the number of histidine-containing peptides bound by the Cu(II)-IMAC columns. This provides an additional possibility for sample simplification in proteomic applications. The number of acylated peptides selected decreased in the order of quaternary amine > N-acetoxysuccinamide > succinic anhydride derivatization. Although the selection of N-terminally derivatized peptides is biased toward peptides that contain more than one histidine, it is not yet possible to predict selectivity. Keywords: Cu(II)-IMAC • peptide acylation • comparative proteomics
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IJS, KILJ, NUK, PNG, UL, UM
Radioresistance represents a major obstacle to a successful outcome for the treatment of non-Hodgkin's lymphoma (NHL). Here we performed a global differential proteome analysis of the mitochondria in ...Raji cells exposed to radiation. The results showed that 23 differentially expressed proteins were identified. Furthermore, GAPDH, RECQL4, MKI67, and ATAD3B could serve as potential biomarkers of radioresistance.
