Bruno M. Alexandre

Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose-phosphate synthase and is required for a proper cold stress response

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here, we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analysing OsCPK17 knockout, silencing and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose-phosphate synthase OsSPS4 and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary disease

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.

Facing challenges in proteomics today and in the coming decade: report of roundtable discussions at the 4th EuPA Scientific Meeting, Portugal, Estoril 2010

Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany FOM Institute for Atomic & Molecular Physics, Science Park 104, 1098 XG Amsterdam, The Netherlands Department of Immunotechnology, Protein Technology, University of Lund, Sweden Agroforestry and Plant Biochemistry and Proteomics Research Group, Department of Biochemistry and Molecular Biology, University of Cordoba, Cordoba, Spain Bioinformatics & High-throughput Analysis Laboratory, Seattle Childrens Research Institute, University of Washington, Seattle, USA Department of Medical Education and Biomedical Informatics, School of Medicine, University of Washington, Seattle, USA Department of Immunotechnology, University of Lund, Sweden Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, Scotland, United Kingdom Institute of Molecular Systems Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Politecnico di Milano, Italy Biomedical Proteomics Group, Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, Geneva, Switzerland Proteomics and Biomarkers, Max Planck Institute of Psychiatry, Kraepelinstr. 2, D-80804 Munich, Germany Division of Molecular Biometry, Department of Medicinal Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden Laboratory of Proteomics, National Institute of Health Dr. Ricardo Jorge, Avenida Padre Cruz, 1649–016 Lisbon, Portugal Division of Hepatology and Gene Therapy, Proteomics Laboratory CIMA, University of Navarra, Avd. Pío XII, 55, 31008 Pamplona, Spain Division of Clinical Protein Science & Imaging, Department of Measurement Technology and Industrial Electrical Engineering, Lund University, Biomedical Center, BMC C13 SE-221 84 Lund, Sweden TCIN, Lloyd Institute, Trinity College Dublin, Dublin, Ireland University of Gothenburg, Krefting Research Centre, Box 424 SE-405 30 Göteborg, Sweden Research Group of Immunology, Hungarian Academy of Sciences ELTE, Pazmany P.s. 1C H-1117 Budapest, Hungary University of Vienna, Department for Molecular Systems Biology, Althanstr. 14, 1090 Vienna, Austria

Proteomics in Detection and Monitoring Chronic Lung Diseases: The Human Nasal Epithelium as a Molecular Model

Asthma and chronic obstructive pulmonary disease (COPD) are major causes of mortality and morbidity worldwide. The current state-of-art diagnosis and management schemes are suboptimal for both diseases as the incidence of asthma has risen by 250% over the last two decades and COPD is estimated to become the third leading cause of death worldwide within the next decade. Additionally, these diseases represent a very important threat to global economies in direct and indirect medical costs and lost working days [1,2]. Asthma is a chronic inflammatory disorder of the airways associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness and coughing. These episodes are usually associated with widespread, but variable, airflow obstruction within the lung [1]. Chronic airflow obstruction is also characteristic of COPD but, in contrast to asthma, is not fully reversible, even under the action of bronchodilators, and is usually progressive. A combination of small airway disease -obstructive bronchiolitis and parenchymal destruction emphysema, leads to COPD clinical manifestation [2]. A number of factors influence a person’s risk of developing these lung diseases, which include host factors, primarily genetic, and environmental factors, such as allergens and tobacco smoke in asthma and COPD, respectively [1-3].

The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications

Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family.

Evening and morning peroxiredoxin-2 redox/oligomeric state changes in obstructive sleep apnea red blood cells: Correlation with polysomnographic and metabolic parameters

We have examined the effects of Obstructive Sleep Apnea (OSA) on red blood cell (RBC) proteome variation at evening/morning day time to uncover new insights into OSA-induced RBC dysfunction that may lead to OSA manifestations. Dysregulated proteins mainly fall in the group of catalytic enzymes, stress response and redox regulators such as peroxiredoxin 2 (PRDX2). Validation assays confirmed that at morning the monomeric/dimeric forms of PRDX2 were more overoxidized in OSA RBC compared to evening samples. Six month of positive airway pressure (PAP) treatment decreased this overoxidation and generated multimeric overoxidized forms associated with chaperone/transduction signaling activity of PRDX2. Morning levels of overoxidized PRDX2 correlated with polysomnographic (PSG)-arousal index and metabolic parameters whereas the evening level of disulfide-linked dimer (associated with peroxidase activity of PRDX2) correlated with PSG parameters. After treatment, morning overoxidized multimer of PRDX2 negatively correlated with fasting glucose and dopamine levels. Overall, these data point toward severe oxidative stress and altered antioxidant homeostasis in OSA RBC occurring mainly at morning time but with consequences till evening. The beneficial effect of PAP involves modulation of the redox/oligomeric state of PRDX2, whose mechanism and associated chaperone/transduction signaling functions deserves further investigation. RBC PRDX2 is a promising candidate biomarker for OSA severity and treatment monitoring, warranting further investigation and validation.

Chronic Obstructive Pulmonary Disease and Proteomics: A Match for Success?

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation that is not fully reversible even under bronchodilators effect, caused by a mixture of small airway disease and parenchymal destruction. COPD is a major cause of morbidity and mortality in adults, and it is now the fourth leading death cause in the world. Cigarette smoking is the main risk factor for COPD but not all smokers will suffer from COPD, suggesting that genetic and other environmental factors are involved in this pathology. Current diagnosis is based on spirometry, but there is recurrent debate on fixed spirometric thresholds in use that lead to misdiagnosis and/or classification of COPD. The available treatments are not effective to reduce or suppress the progression of COPD. Hence, there is an urgent need to better understand the molecular mechanisms of COPD pathogenesis to provide clinicians with reliable diagnosis and treatment tools for COPD. Proteomics, defined by the comprehensive study of the proteome, has the potential to respond to this need by providing protein profiles of a particular disease and, at the same time, by identifying specific biomarkers that can be used to better understand, diagnose and manage the disease. Here, we shortly review COPD history and pathology and how proteomics can match COPD for success.

Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue.

Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK.

Evening and morning alterations in Obstructive Sleep Apnea red blood cell proteome

This article presents proteomics data referenced in [1] Using proteomics-based evaluation of red blood cells (RBCs), we have identified differentially abundant proteins associated with Obstructive Sleep Apnea Syndrome (OSA). RBCs were collected from peripheral blood of patients with moderate/severe OSA or snoring at pre- (evening) and post-night (morning) polysomnography, so that proteome variations between these time points could be assessed. RBC cytoplasmic fraction depleted of hemoglobin, using Hemovoid™ system, were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), the 2D image software-based analyzed and relevant differentially abundant proteins identified by mass spectrometry (MS). MS identified 31 protein spots differentially abundant corresponding to 21 unique proteins possibly due to the existence of post-translational modification regulations. Functional analysis by bioinformatics tools indicated that most proteins are associated with catalytic, oxidoreductase, peroxidase, hydrolase, ATPase and anti-oxidant activity. At morning a larger numbers of differential proteins including response to chemical stimulus, oxidation reduction, regulation of catalytic activity and response to stress were observed in OSA. The data might support further research in OSA biomarker discovery and validation.