Heat shock protein 90 is downregulated in calcific aortic disease

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Sputum Vitamin D Binding Protein (VDBP) GC1S/1S Genotype Predicts Airway Obstruction : A Prospective Study in Smokers with COPD

Introduction: The vitamin D binding protein (VDBP, also known as GC-globulin) and vitamin D deficiency have been associated with chronic obstructive pulmonary disease (COPD). rs7041 and rs4588 are two single nucleotide polymorphisms of the VDBP gene, including three common allelic variants (GC1S, GC1F and GC2). Previous studies primarily assessed the serum levels of vitamin D and VDBP in COPD. However, less is known regarding the impact of the local release of VDBP on COPD lung function. Thus, we examined the association of sputum and plasma VDBP with lung function at baseline and at four years, and examined potential genetic polymorphism interactions. Methods: The baseline levels of sputum VDBP, plasma VDBP and plasma 25-OH vitamin D, as well as the GC rs4588 and rs7041 genotypes, were assessed in a 4-year Finnish follow-up cohort (n = 233) of non-smokers, and smokers with and without COPD. The associations between the VDBP levels and the longitudinal decline of lung function were further analysed. Results: High frequencies of the haplotypes in rs7041/rs4588 were homozygous GC1S/1S (42.5%). Higher sputum VDBP levels in stage I and stage II COPD were observed only in carriers with GC1S/1S genotype when compared with non-smokers (p = 0.034 and p = 0.002, respectively). Genotype multivariate regression analysis indicated that the baseline sputum VDBP and FEV1/FVC ratio at baseline independently predicted FEV1% at follow-up. Discussion and Conclusion: The baseline sputum VDBP expression was elevated in smokers with COPD among individuals with the GC1S/1S genotype, and predicted follow-up airway obstruction. Our results suggest that the GC polymorphism should be considered when exploring the potential of VDBP as a biomarker for COPD.Peer reviewe

Hemoglobin alpha and beta are ubiquitous in the human lung, decline in idiopathic pulmonary fibrosis but not in COPD

Background: Idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are disorders of the lung parenchyma. They share the common denominators of a progressive nature and poor prognosis. The goal was to use non-biased proteomics to discover new markers for these diseases. Methods: Proteomics of fibrotic vs. control lung tissue suggested decreased levels of several spots in the lung specimens of IPF patients, which were identified as Hemoglobin (Hb) alpha and beta monomers and Hb alpha complexes. The Hb alpha and beta monomers and complexes were investigated in more detail in normal lung and lung specimens of patients with IPF and COPD by immunohistochemistry, morphometry and mass spectrometry (MS). Results: Both Hb monomers, in normal lung, were expressed especially in the alveolar epithelium. Levels of Hb alpha and beta monomers and complexes were reduced/lost in IPF but not in the COPD lungs when compared to control lung. MS-analyses revealed Hb alpha modification at cysteine105 (Cys alpha 105), preventing formation of the Hb alpha complexes in the IPF lungs. Hb alpha and Hb beta were expressed as complexes and monomers in the lung tissues, but were secreted into the bronchoalveolar lavage fluid and/or induced sputum supernatants as complexes corresponding to the molecular weight of the Hb tetramer. Conclusions: The abundant expression of the oxygen carrier molecule Hb in the normal lung epithelium and its decline in IPF lung are new findings. The loss of Hb complex formation in IPF warrants further studies and may be considered as a disease-specific modification

Heat shock protein 90 is downregulated in calcific aortic valve disease

Abstract Background Calcific aortic valve disease (CAVD) is an atheroinflammatory process; finally it leads to progressive calcification of the valve. There is no effective pharmacological treatment for CAVD and many of the underlying molecular mechanisms remain unknown. We conducted a proteomic study to reveal novel factors associated with CAVD. Methods We compared aortic valves from patients undergoing valvular replacement surgery due to non-calcified aortic insufficiency (control group, n = 5) to a stenotic group (n = 7) using two-dimensional difference gel electrophoresis (2D-DIGE). Protein spots were identified with mass spectrometry. Western blot and immunohistochemistry were used to validate the results in a separate patient cohort and Ingenuity Pathway Analysis (IPA) was exploited to predict the regulatory network of CAVD. Results We detected an upregulation of complement 9 (C9), serum amyloid P-component (APCS) and transgelin as well as downregulation of heat shock protein (HSP90), protein disulfide isomerase A3 (PDIA3), annexin A2 (ANXA2) and galectin-1 in patients with aortic valve stenosis. The decreased protein expression of HSP90 was confirmed with Western blot. Conclusions We describe here a novel data set of proteomic changes associated with CAVD, including downregulation of the pro-inflammatory cytosolic protein, HSP90

NO-mediated apoptosis in yeast

Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in biological systems, among them the control of the apoptotic signalling cascade. By combining proteomic, genetic and biochemical approaches we demonstrate that NO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are crucial mediators of yeast apoptosis. Using indirect methodologies and a NO-selective electrode, we present results showing that H2O2-induced apoptotic cells synthesize NO that is associated to a nitric oxide synthase (NOS)-like activity as demonstrated by the use of a classical NOS kit assay. Additionally, our results show that yeast GAPDH is a target of extensive proteolysis upon H2O2-induced apoptosis and undergoes S-nitrosation. Blockage of NO synthesis with Nomega-nitro-L-arginine methyl ester leads to a decrease of GAPDH S-nitrosation and of intracellular reactive oxygen species (ROS) accumulation, increasing survival. These results indicate that NO signalling and GAPDH S-nitrosation are linked with H2O2-induced apoptotic cell death. Evidence is presented showing that NO and GAPDH S-nitrosation also mediate cell death during chronological life span pointing to a physiological role of NO in yeast apoptosis.This work was supported by a grant from FCT-Fundação para a Ciência e a Tecnologia (POCI/BIA-BCM/57364/2004). B.A. has a fellowship from FCT (SFRH/BD/15317/2005). We are also grateful to FWF for a Lipotox grant to F.M. and B.M. and for grant no. S-9304-B05 to F.M. and S.B

Human placental proteomics and exon variant studies link AAT/SERPINA1 with spontaneous preterm birth

Background: Preterm birth is defined as live birth before 37 completed weeks of pregnancy, and it is a major problem worldwide. The molecular mechanisms that lead to onset of spontaneous preterm birth are incompletely understood. Prediction and evaluation of the risk of preterm birth is challenging as there is a lack of accurate biomarkers. In this study, our aim was to identify placental proteins that associate with spontaneous preterm birth. Methods: We analyzed the proteomes from placentas to identify proteins that associate with both gestational age and spontaneous labor. Next, rare and potentially damaging gene variants of the identified protein candidates were sought for from our whole exome sequencing data. Further experiments we performed on placental samples and placenta-associated cells to explore the location and function of the spontaneous preterm labor-associated proteins in placentas. Results: Exome sequencing data revealed rare damaging variants in SERPINA1 in families with recurrent spontaneous preterm deliveries. Protein and mRNA levels of alpha-1 antitrypsin/SERPINA1 from the maternal side of the placenta were downregulated in spontaneous preterm births. Alpha-1 antitrypsin was expressed by villous trophoblasts in the placenta, and immunoelectron microscopy showed localization in decidual fibrinoid deposits in association with specific extracellular proteins. siRNA knockdown in trophoblast-derived HTR8/SVneo cells revealed that SERPINA1 had a marked effect on regulation of the actin cytoskeleton pathway, Slit–Robo signaling, and extracellular matrix organization. Conclusions: Alpha-1 antitrypsin is a protease inhibitor. We propose that loss of the protease inhibition effects of alpha-1 antitrypsin renders structures critical to maintaining pregnancy susceptible to proteases and inflammatory activation. This may lead to spontaneous premature birth.publishedVersionPeer reviewe

Expression of CPPED1 in human trophoblasts is associated with timing of term birth

Understanding of timing of human parturition is incomplete. Therefore, we carried out proteomic analyses of full-term placentas from uncomplicated pregnancies to identify protein signatures associated with the onset of spontaneous delivery. We found quantitative associations of 10 proteins with spontaneous term birth, evident either in the basal or in the chorionic plates or in both. Additional 18 proteins were associated according to the location within placenta indicating local variations in protein amounts. Calcineurin-like phosphoesterase domain-containing 1 (CPPED1), a phosphatase previously suggested dephosphorylating AKT1/PKB, was one of the identified proteins. qRT-PCR revealed the mRNA level of CPPED1 was higher in elective caesarean deliveries than in spontaneous births, while immunohistochemistry showed CPPED1 in cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts. Noteworthy, phosphorylation status of AKT1 did not differ between placentas from elective caesarean and spontaneous deliveries. Additionally, analyses of samples from infants indicated that single-nucleotide polymorphisms rs11643593 and rs8048866 of CPPED1 were associated with duration of term pregnancy. Finally, post-transcriptional silencing of CPPED1 in cultured HTR8/SVneo cells by siRNAs affected gene expression in pathways associated with inflammation and blood vessel development. We postulate that functions regulated by CPPED1 in trophoblasts at choriodecidual interphase have a role in the induction of term labour, but it may be independent of AKT1

A novel human leiomyoma tissue derived matrix for cell culture studies

Background: The composition of the matrix molecules is important in in vitro cell culture experiments of e.g. human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived products, such as Matrigel (R), are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel (R) is non-human in origin, its molecular composition does not accurately simulate human TME. We have previously described a solid 3D organotypic myoma disc invasion assay, which is derived from human uterus benign leiomyoma tumor. Here, we describe the preparation and analyses of a processed, gelatinous leiomyoma matrix, named Myogel. Methods: A total protein extract, Myogel, was formulated from myoma. The protein contents of Myogel were characterized and its composition and properties compared with a commercial mouse Matrigel (R). Myogel was tested and compared to Matrigel (R) in human cell adhesion, migration, invasion, colony formation, spheroid culture and vessel formation experiments, as well as in a 3D hanging drop video image analysis. Results: We demonstrated that only 34 % of Myogel's molecular content was similar to Matrigel (R). All test results showed that Myogel was comparable with Matrigel (R), and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel (R) in in vitro Transwell (R) invasion and capillary formation assays. Conclusions: In conclusion, we have developed a novel Myogel TME matrix, which is recommended for in vitro human cell culture experiments since it closely mimics the human tumor microenvironment of solid cancers.Peer reviewe

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement Nu 241500 (BuruliVac), from Fundacao Calouste Gulbenkian and from Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). JBG, TGM and AGF had a personal grant from the Portuguese Science and Technology Foundation (FCT) (SFRH/BD/33573/2009, SFRH/BD/41598/2007 and SFRH/BPD/68547/2010, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript