Proteomic changes related to actin cytoskeleton function in the skin of vildagliptin-treated mice.

BACKGROUND: Vildagliptin, a dipeptidyl peptidase-4 inhibitor (DPP-4i) is a widely used type 2 diabetes medication that is associated with an up-to 10-fold increased risk for the development of bullous pemphigoid (BP), an autoimmune skin disease. The mechanism by which vildagliptin promotes the development of BP remains unknown. OBJECTIVE: To elucidate effects of vildagliptin treatment on the mouse cutaneous proteome. METHODS: We analyzed the cutaneous proteome of nondiabetic mice treated for 12 weeks with vildagliptin using label-free shotgun mass spectrometry (MS), two-dimensional difference gel electrophoresis (2D-DIGE), immunohistochemistry, immunoblotting, and quantitative real-time polymerase chain reaction. RESULTS: Although vildagliptin treatment did not cause any clinical signs or histological changes in the skin, separate MS and 2D-DIGE analyses revealed altered cutaneous expression of several proteins, many of which were related to actin cytoskeleton remodeling. Altogether 18 proteins were increased and 40 were decreased in the vildagliptin-treated mouse skin. Both methods revealed increased levels of beta-actin and C->U-editing enzyme APOBEC2 in vildagliptin-treated mice. However, elevated levels of a specific moesin variant in vildagliptin-treated animals were only detected with 2D-DIGE. Immunohistochemical staining showed altered cutaneous expression of DPP-4, moesin, and galectin-1. The changed proteins detected by MS and 2D-DIGE were linked to actin cytoskeleton remodeling, transport, cell movement and organelle assembly. CONCLUSION: Vildagliptin treatment alters the cutaneous proteome of nondiabetic mice even without clinical signs in the skin. Cytoskeletal changes in the presence of other triggering factors may provoke a break of immune tolerance and further promote the development of BP.

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.

Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease.

BACKGROUND: FINCA disease is a pediatric cerebropulmonary disease caused by variants in the NHL repeat-containing 2 (NHLRC2) gene. Neurological symptoms are among the first manifestations of FINCA disease, but the consequences of NHLRC2 deficiency in the central nervous system are currently unexplored. METHODS: The orthologous mouse gene is essential for development, and its complete loss leads to early embryonic lethality. In the current study, we used CRISPR/Cas9 to generate an Nhlrc2 knockin (KI) mouse line, harboring the FINCA patient missense mutation (c.442G > T, p.Asp148Tyr). A FINCA mouse model, resembling the compound heterozygote genotype of FINCA patients, was obtained by crossing the KI and Nhlrc2 knockout mouse lines. To reveal NHLRC2-interacting proteins in developing neurons, we compared cortical neuronal precursor cells of E13.5 FINCA and wild-type mouse embryos by two-dimensional difference gel electrophoresis. RESULTS: Despite the significant decrease in NHLRC2, the mice did not develop severe early onset multiorgan disease in either sex. We discovered 19 altered proteins in FINCA neuronal precursor cells; several of which are involved in vesicular transport pathways and actin dynamics which have been previously reported in other cell types including human to have an association with dysfunctional NHLRC2. Interestingly, isoform C2 of hnRNP C1/C2 was significantly increased in both developing neurons and the hippocampus of adult female FINCA mice, connecting NHLRC2 dysfunction with accumulation of RNA binding protein. CONCLUSIONS: We describe here the first NHLRC2-deficient mouse model to overcome embryonic lethality, enabling further studies on predisposing and causative mechanisms behind FINCA disease. Our novel findings suggest that disrupted RNA metabolism may contribute to the neurodegeneration observed in FINCA patients.

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.

Heat shock protein 90 is downregulated in calcific aortic valve disease.

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.

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.

An approach to comprehensive genome and proteome expression analyses in Schwann cells and neurons during peripheral nerve myelin formation.

Peripheral nerve myelination is a complex event resulting from spatially and temporally regulated reciprocal interactions between the neuron and myelin-forming Schwann cells. The dynamic process and the protein functional modules and networks that operate throughout the myelination process are poorly understood because of a lack of methodologies suitable for observing specific changes in the Schwann cell/neuron-unit. The identification of the precise roles for the proteins participating in the functional modules and networks that participate in the myelination process is hindered by the cellular and molecular complexity of the nervous tissue itself. We have developed an approach based on a myelinating dorsal root ganglion explant model that allows distinguishing clear, reproducible and predictable differences between the biochemical properties and the genomic and proteomic expression profiles of both cellular components of the Schwann cell/neuron unit at different stages of the myelination process. This model, derived from E13.5 C57BL/6J mouse embryos, is sufficiently robust for use in identifying the protein functional networks and modules related to peripheral nerve myelin formation. The genomic expression profiles of the selected neuronal, Schwann cell and myelin-specific proteins in the cultures reflect in vivo profiles reported in the literature, and the structural and ultrastructural properties of the myelin, as well as the myelination schedule of the cultures, closely resemble those observed in peripheral nerves in situ. The RNA expression data set is available through NCBI gene expression omnibus accession GSE60345. We have developed a reproducible and robust cell culture-based approach, accompanied by a genome-wide expression data set, which allows studying myelination in the peripheral nervous system at the proteomic and transcriptomic levels in Schwann cells and neurons. Myelinating dorsal root explant cultures, prepared from C57BL/6J mouse embryos, present distinct developmental stages comparable to those observed in a peripheral nerve in situ. This model can be used for identifying the protein functional networks and modules related to peripheral nerve myelin formation.

Stabilin-1 expression defines a subset of macrophages that mediate tissue homeostasis and prevent fibrosis in chronic liver injury.

Macrophages are key regulators of fibrosis development and resolution. Elucidating the mechanisms by which they mediate this process is crucial for establishing their therapeutic potential. Here, we use experimental models of liver fibrosis to show that deficiency of the scavenger receptor, stabilin-1, exacerbates fibrosis and delays resolution during the recovery phase. We detected a subset of stabilin-1(+) macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL (MDA-LDL) uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP(+) fibrogenic cells. Stabilin-1(-/-) macrophages demonstrated a proinflammatory phenotype during liver injury and the normal induction of Ly6C(lo) monocytes during resolution was absent in stabilin-1 knockouts leading to persistence of fibrosis. Human stabilin-1(+) monocytes efficiently internalized MDA-LDL and this suppressed their ability to secrete CCL3, suggesting that loss of stabilin-1 removes a brake to CCL3 secretion. Experiments with cell-lineage-specific knockouts revealed that stabilin-1 expression in myeloid cells is required for the induction of this subset of macrophages and that increased fibrosis occurs in their absence. This study demonstrates a previously unidentified regulatory pathway in fibrogenesis in which a macrophage scavenger receptor protects against organ fibrosis by removing fibrogenic products of lipid peroxidation. Thus, stabilin-1(+) macrophages shape the tissue microenvironment during liver injury and healing.

Dual targeted poplar ferredoxin NADP(+) oxidoreductase interacts with hemoglobin 1.

Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar (Populus tremula × tremuloides) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast (Saccharomyces cerevisiae) Δyhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP(+) oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast Δyhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol.

Lung tissue proteomics identifies elevated transglutaminase 2 levels in stable chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by irreversible airflow limitation. Cigarette smoking represents the main risk factor, but the specific mechanisms of COPD are not completely understood. Our aim was to identify COPD-specific proteomic changes involved in disease onset and severity. A comparative proteomic analysis of 51 lung tissues from nonsmokers, smokers, smokers with mild to moderate (stage I-II) COPD, severe to very severe COPD (stage III-IV), and patients with α-1-antitrypsin deficiency (AATD) and idiopathic pulmonary fibrosis (IPF) was performed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Selected COPD-specific changes were validated by immunoblotting and further by ELISA in 120 induced sputum and plasma samples from nonsmokers, smokers, and patients with COPD (stage I-III). Altogether 82 altered proteins were identified comprising COPD-, AATD-, and IPF-specific, overlapping, and unspecific changes. Cathepsin D (CTSD), dihydropyrimidinase-related protein 2 (DPYSL2), transglutaminase 2 (TGM2), and tripeptidyl-peptidase 1 (TPP1) were validated as COPD-specific. TGM2 was not associated with smoking and correlated with COPD severity in lung tissue. TGM2 levels in sputum and plasma were elevated in patients with COPD (stage II-III) and correlated with lung function. In conclusion, new proteins related to COPD onset and severity could be identified with TGM2 being a novel potential diagnostic and therapeutic target for COPD. Further studies in carefully characterized cohorts are required to validate the identified changes.

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®, are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel® 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®. Myogel was tested and compared to Matrigel® 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®. All test results showed that Myogel was comparable with Matrigel®, and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel® in in vitro Transwell® 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.

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.

Analysis of molecular changes after autologous cell therapy in swine myocardial infarction tissue can reveal novel targets for future therapy.

Although several studies have demonstrated a functional recovery of infarcted myocardial tissue after cell therapy, little is known about the molecular mechanisms behind it. The aim of this study was to characterize the effect of cell therapy at the molecular level to screen for novel target candidates for future therapy of infarcted myocardial tissue. We used a swine acute myocardial infarction model evoked by transient occlusion of the circumflex coronary artery. Autologous bone marrow-derived mononuclear cells (BMMCs) or saline were injected intramyocardially or into the circumflex coronary artery. Samples for protein and RNA analysis were collected from the infarction area and healthy myocardium after a 3 week recovery period and analysed by two-dimensional gel electrophoresis (2DE) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Proteomic screening detected 13 protein spots which were altered after infarction but had been restored by BMMC treatment. The identification of seven proteins by mass spectrometry revealed that five proteins with decreased expression after infarction corresponded to mitochondrial proteins involved in energy metabolism. Their restored levels after BMMC treatment indicate their involvement in the recovery of heart function. In contrast, the elevated levels of α-crystallin B chain and cathepsin D after infarction suggest an involvement in the pathological mechanisms causing a decreased heart function. This study reveals that cell therapy with BMMCs after myocardial infarction causes restoration of several altered protein levels after 3 weeks and identifies potential marker proteins involved in the pathology of infarction.

Characterization of chronic subdural hematoma fluid proteome.

BACKGROUND: Chronic subdural hematoma (CSDH) is commonly caused by head injury, but the progression of CSDH is recognized as an inflammatory process. The protein composition of the CSDH fluid has not been fully elucidated, nor has the contribution of its components to the enlargement of the hematoma cavity and to its chronic manifestation. OBJECTIVE: To characterize the protein content of CSDH fluid and study the differences between CSDH fluid and serum to identify proteins putatively involved in the pathogenesis of CSDH. METHODS: CSDH fluid and serum of 5 patients were investigated with 2-dimensional gel electrophoresis followed by glycosylation-specific fluorescence staining and mass spectrometry. RESULTS: Two-dimensional gel electrophoresis revealed approximately 1100 protein spots in the CSDH fluid. We identified 213 spots representing 57 different proteins, most of which were glycosylated. The comparison with serum revealed 11 proteins with elevated levels in the CSDH fluid including carbonic anhydrase I, catalase, ferritin light chain, fibrinogen (α, ß, γ), hemoglobin (α, ß), malate dehydrogenase, peroxiredoxin 2, and transforming growth factor-ß-induced protein ig-h3. The levels of haptoglobin and a fragment of complement C4 were decreased. Changes in spot positions were detected for apolipoprotein A1 and a fragment of complement C3. CONCLUSION: The hematoma fluid originates mainly from blood and the results suggest the involvement of coagulation and fibrinolysis cascades. However, proteins with a potential role in CSDH pathogenesis were detected including carbonic anhydrase I, transforming growth factor-ß-induced protein ig-h3, and the altered components of the complement system. Inflammation and fibrosis indicate targets for further studies in the pathogenesis of CSDH.

Distribution and levels of alpha-1-antitrypsin in the lung and plasma in smokers and chronic obstructive pulmonary disease.

Our recent non-biased proteomic screening study revealed elevated SerpinA1 i.e. alpha-1-antitrypsin (AAT) levels in induced sputum of smokers with Chronic obstructive pulmonary disease (COPD). This study was designed to further investigate the role of AAT in smokers and subjects with COPD. The expression/distribution of AAT was studied by immunohistochemistry/digital image morphometry in the lung, by Western blot in the lung and sputum, and by ELISA in the plasma at baseline (n = 349) and after a 2-year follow-up (n = 58). AAT was localized mainly in airway and alveolar epithelium and endothelium, especially in smokers and in those with COPD. AAT was elevated in smokers and in subjects with COPD in the lung endothelial cells. Total lung AAT immunoreactivity was elevated in subjects with moderate COPD compared with smokers and with non-smokers. AAT showed elevated tendency in sputum of smokers with COPD compared with 'healthy' smokers. Plasma AAT levels were elevated in smokers with/without COPD compared with non-smokers. In the follow-up, plasma AAT concentrations decreased significantly after quitting smoking. Chronic smoking/COPD leads to AAT elevation especially in the endothelium of the lung periphery; these changes reflect only modestly to the AAT in sputum, while plasma AAT significantly reflects smoking-related systemic manifestations, and decreases after smoking cessation.

Sputum proteomics identifies elevated PIGR levels in smokers and mild-to-moderate COPD.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. However, the exact mechanisms leading to COPD and its progression are still poorly understood. In this study, induced sputum was analyzed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry to identify proteins involved in COPD pathogenesis. The comparison of nonsmokers, smokers, and smokers with moderate COPD revealed 15 changed proteins with the majority, including polymeric immunoglobulin receptor (PIGR), being elevated in smokers and subjects with COPD. PIGR, which is involved in specific immune defense and inflammation, was further studied in sputum, lung tissue, and plasma by Western blot, immunohistochemistry/image analysis, and/or ELISA. Sputum PIGR was characterized as glycosylated secretory component (SC). Lung PIGR was significantly elevated in the bronchial and alveolar epithelium of smokers and further increased in the alveolar area in mild to moderate COPD. Plasma PIGR was elevated in smokers and smokers with COPD compared to nonsmokers with significant correlation to obstruction. In conclusion, new proteins in smoking-related chronic inflammation and COPD could be identified, with SC/PIGR being one of the most prominent not only in the lung but also in circulating blood.

Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture.

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity.

Proteomic studies on receptor for advanced glycation end product variants in idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease.

PURPOSE: Proteomic screening revealed declined levels of the receptor for advanced glycation end products (RAGE) in human idiopathic pulmonary fibrosis (IPF). This study was undertaken to investigate the different RAGE isoforms in two lung diseases with destruction of the lung parenchyma, i.e. IPF and chronic obstructive pulmonary disease (COPD). EXPERIMENTAL DESIGN: RAGE was analyzed by 2-DE, MS and Western blotting using lung tissues from non-smokers, smokers, patients with IPF, COPD and α-1-antitrypsin deficiency (AAT) and by ELISA from the bronchoalveolar lavage fluid samples. RESULTS: RAGE, detected by 2-DE in the control lung, was confirmed to be glycosylated, soluble, C-truncated RAGE with characteristics indicative of the presence of endogenous secretory RAGE (esRAGE). Further studies revealed a decrease of the full length-RAGE (FL-RAGE) and its C-terminal processed variant (cRAGE) in the lung tissues of IPF and COPD patients but not in AAT. The esRAGE level was reduced in IPF but was unchanged in COPD. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows an involvement of the three RAGE variants (FL-RAGE, cRAGE, esRAGE) in IPF. The decline of FL-RAGE and cRAGE, but not esRAGE, in COPD lungs is evidence of involvement of specific RAGE variants also in this disease.

Hemoglobin α and ß 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) α and ß monomers and Hbα complexes. The Hbα and ß 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 Hbα and ß monomers and complexes were reduced/lost in IPF but not in the COPD lungs when compared to control lung. MS-analyses revealed Hbα modification at cysteine105 (Cysα105), preventing formation of the Hbα complexes in the IPF lungs. Hbα and Hbß 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.

Protein phosphorylation in mitochondria --a study on fermentative and respiratory growth of Saccharomyces cerevisiae.

Phosphorylation as a posttranslational protein modification is a common subject of proteomic studies, but phosphorylation in mitochondria is still poorly investigated. The study presented here applied 2-DE to characterize phosphorylation in the yeast mitochondrial proteome and identified 59 spots corresponding to 34 phosphorylated mitochondrial or mitochondria-associated proteins. Most of these proteins presented putative substrates of mitogen-activated protein and target of rapamycin kinases, cAMP-dependent protein kinase, cyclin-dependent kinases and Snf1p suggesting them as key players in the phosphorylation of mitochondrial or mitochondria-associated proteins. The dynamic behaviour of the phosphoproteome under a major metabolic change, the shift from fermentation to respiration (diauxic shift), was further studied. Eight proteins (Ald4p, Eft1p/2p, Eno1p, Eno2p, Om14p, Pda1p, Qcr2p, Sdh1p) had growth dependent changes in their phosphorylation, indicating a role of phosphorylation-dependent regulation of translation, metabolic pathways (e.g. glucose fermentation, tricarboxylic acid cycle, pyruvate dehydrogenase and its bypass) and respiratory chain.