Tânia Simões

Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathies

α-Synuclein misfolding and aggregation is a hallmark in Parkinsons disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.

Occupational Exposure to Environmental Tobacco Smoke: A Study in Lisbon Restaurants

Environmental tobacco smoke (ETS), also referred to as secondhand smoke (SHS), is a major threat to public health and is increasingly recognized as an occupational hazard to workers in the hospitality industry. Therefore, several countries have implemented smoke-free regulations at hospitality industry sites. In Portugal, since 2008, legislation partially banned smoking in restaurants and bars but until now no data have been made available on levels of indoor ETS pollution/exposure at these locations. The aim of this study was to examine the occupational exposure to ETS/SHS in several restaurants in Lisbon, measured by indoor fine particles (PM2.5) and urinary cotinine concentration in workers, after the partial smoking ban in Portugal. Results showed that the PM2.5 median level in smoking designated areas was 253 μg/m3, eightfold higher than levels recorded in canteens or outdoor. The nonsmoking rooms of mixed restaurants exhibited PM2.5 median level of 88 μg/m3, which is higher than all smoke-free locations studied, approximately threefold greater than those found in canteens. Importantly, urinary cotinine concentrations were significantly higher in nonsmoker employees working in those smoking designated areas, confirming exposure to ETS. The proportion of smokers in those rooms was found to be significantly positively correlated with nonsmoker urinary cotinine and indoor PM2.5 levels, establishing that both markers were occupational-ETS derived. The use of reinforced ventilation systems seemed not to be sufficient to decrease the observed ETS pollution/exposure in those smoking locations. Taken together, these findings demonstrate that the partial restrictions on smoking in Portuguese venues failed to provide adequate protection to their employees, irrespective of protective measures used. Therefore, a smoke-free legislation protecting individuals from exposure to ETS/SHS in all public places and workplaces is urgently needed in Portugal.

Heat-mediated enrichment of α-synuclein from cells and tissue for assessing post-translational modifications

α‐synuclein (α‐syn) is the major component of Lewy bodies, a pathological hallmark of Parkinsons disease and other synucleinopathies. The characterization of α‐syn post‐translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward α‐syn PTMs. Here, by taking advantage of α‐syn thermostability, we applied a method to achieve high enrichment of soluble α‐syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% α‐syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N‐terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of α‐syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies.

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

A possible approach for gel-based proteomic studies in recalcitrant woody plants

Woody plants are particularly difficult to investigate due to high phenolic, resin, and tannin contents and laborious sample preparation. In particular, protein isolation from woody plants for two-dimensional gel electrophoresis (2-DE) is challenging as secondary metabolites negatively interfere with protein extraction and separation. In this study, three protein extraction protocols, using TCA, phenol and ethanol as precipitation or extraction agents, were tested in order to select the more efficient for woody recalcitrant plant gel-based proteomics. Grapevine leaves, pine needles and cork oak ectomycorrhizal roots were used to represent woody plant species and tissues. The phenol protocol produced higher quality 2-DE gels, with increased number of resolved spots, better spot focusing and representation of all molecular mass and isoelectric point ranges tested. In order to test the compatibility of the phenol extracted proteomes with protein identification several spots were excised from the phenol gels and analyzed by mass spectrometry (MALDI-TOF/TOF). Regardless the incomplete genome/protein databases for the plant species under analysis, 49 proteins were identified by Peptide Mass Fingerprint (PMF). Proteomic data have been deposited to the ProteomeXchange with identifier PXD000224. Our results demonstrate the complexity of protein extraction from woody plant tissues and the suitability of the phenol protocol for obtaining high quality protein extracts for efficient 2-DE separation and downstream applications such as protein identification by mass spectrometry.

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].