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Publications (109)
In this study, we examined the effect of six months of positive airway pressure (PAP) therapy on Obstructive Sleep Apnea (OSA) red blood cell (RBC) proteome by two dimensional difference gel electrophoresis (2D-DIGE) - based proteomics followed by Western blotting (WB) validation. The discovered dysregulated proteins/proteoforms are associated with...
Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earlies...
ABSTRACT
Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine.
Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect th...
Introduction: Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide and associated with decreased lung function and inflammation. The heterogeneity of COPD and its molecular and clinical features hinder efficient patient stratification and introduction of personalized therapeutic approaches. The available clin...
Background: Obstructive sleep apnea (OSA) is considered as metabolic syndrome, which diagnosis in early stages is challenging. Recent studies found relation between transthyretin (TTR) protein modifications present in human plasma samples and sleep disorders1,2. Mass Spectrometric Immunoassay (MSIA) was successfully applied previously on identifica...
Introduction:
Obstructive sleep apnea syndrome (OSAS) is associated with cardiovascular/metabolic complications. Some analytical parameters (homocysteine, glycemic and lipidic profiles) are recognized markers of these consequences. Limited data is available on the association of these markers and OSAS's severity/response to positive airway pressur...
α-Synuclein misfolding and aggregation is a hallmark in Parkinson's 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...
Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age- associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mech...
Author summary
Parkinson disease is an age-associated neurodegenerative disorder characterized by the loss of dopamine-producing neurons from a region in the brain known as the substantia nigra and by the accumulation of the protein alpha-synuclein in intracellular clumps called inclusions. Whether these inclusions are the cause or a consequence of...
Variation of aSyn acetylation levels with ageing.
Brain protein extracts of WT and T2.KO young (2 month) and old (8 month) mice were probed for acetyl-lysine (red) and aSyn (green) (n = 4 per group). The ratio of acetyl-lysine/aSyn ratio is presented. **p < 0.01, **** p < 0.0001, ordinary one-way ANOVA followed by Tukey’s multiple comparisons test....
Acetylation-resistant aSyn mutant maintains the typical NMR signal and membrane-binding ability.
(A) Superposition of 2D 1H-15N HSQC NMR spectra of recombinant 15N-labelled aSyn WT (black), K6+10R (red). (D) Residue-specific changes in 1H-15N HSQC signal intensities of aSyn WT (black) and aSyn K6+10R (red) upon addition of SUVs formed by POPC:POPA...
Acetylation-resistant mutant of aSyn is toxic.
Lactate dehydrogenase levels (LDH) were measured in the supernatants of primary cultures infected with AVV6 encoding for WT, KQ or KR aSyn, at different time points after transduction. The KR mutant is toxic 3 days after infection, and the toxicity is then indistinguishable at later time points. Data i...
Expression of WT aSyn in the SN is toxic over time.
(A) Brain sections immunostained for TH (red panels) and aSyn (Syn-1) (green panels) 1, 2 and 3 weeks after injection with vectors encoding for EGFP or WT aSyn. Scale bar for isolated channels 200 μm and for merged channels 100 μm. (B) Stereological counting of the number of TH-positive neurons in...
Aggregation pattern of aSyn in the rat substantia nigra.
Brain sections immunostained for aggregated-aSyn (red) and GFP or pS129 aSyn (green) from representative animals 3 weeks after injection with AAV6 vectors encoding for GFP and WT, KR or KQ aSyn. (A) 5G4 and GFP (GFP group) or pS129 (aSyn groups) merged signal with DAPI is presented. Scale bar...
Raw data.
Results from Figs 1F, 2B, 2F, 3A, 3B, 4A, 4B, 4C, 5C, 6B, 7B, 7E, 7F, S1, S4, S6 and S7B.
(XLSX)
aSyn KxK motifs are less susceptible to chemical acetylation in vitro.
Peptide mass spectrometry analysis of chemically acetylated recombinant aSyn, showing the number of acetylation occurrences. Each green bar represents a detected peptide, and a red dash indicates an acetylation.
(TIF)
aSyn acetylation in mouse brain, identified by peptide mass fingerprint (PMF) (trypsin digestion).
Theoretical peptide mass (Da); error (ppm); Start-end identified peptides; peptide sequences; putative acetylation residues. Oxid (M), N-terminal acetylation and acetylation (K) as variable modifications.
(DOCX)
Autophagy is impaired upon T2.KD in the cell model of aSyn aggregation.
Protein extracts from Scr or T2.KD cells co-transfected with SynT and Synphilin-1 were probed for P62 and β-Actin. Normalized levels of P62 are presented (n = 3). ** p < 0.01, unpaired t-test with equal SD. Data in S1 Data.xls.
(TIF)
aSyn constructs, viral vector details, and experimental paradigm.
(A) aSyn double mutants mimicking the acetylated (KQ) or the acetylation-resistant (KR) variants of aSyn on K6 and K10. (B) Recombinant adeno-associated viral vectors (AAV) serotype 6 expressing green fluorescent protein (GFP), human wild type (WT), KQ, KR variants of aSyn under the...
Obstructive sleep apnea syndrome (OSAS) is a systemic inflammatory disease associated with cardiovascular consequences. Red blood cell distribution width (RDW), mean platelet volume (MPV), and platelet distribution width (PDW) are recognized biomarkers of cardiovascular morbidity/mortality. Limited data is available on the association between these...
This article presents proteomics data referenced in [1] (A. Feliciano, F. Vaz, V.M. Torres, C. Valentim-Coelho, R. Silva, V. Prosinecki , B. Alexandre, A.S. Carvalho, R. Matthiesen, A. Malhotra, P. Pinto, C. Bárbara, D. Penque, 2016). Using proteomics-based evaluation of red blood cells (RBC), we have identified differentially abundant proteins ass...
Uncovering unknown pathological mechanisms and body response to applied medication are the driving forces toward personalized medicine. In this post-genomic era, all eyes are turned to the proteomics fi eld, searching for answers and explanations by investigating the gene end point functional units—proteins and their proteoforms. The development of...
Uncovering unknown pathological mechanisms and body response to applied medication are the driving forces toward personalized medicine. In this post-genomic era, all eyes are turned to the proteomics fi eld, searching for answers and explanations by investigating the gene end point functional units—proteins and their proteoforms. The development of...
The quest to understand biological systems requires further attention of the scientific community to the challenges faced in proteomics. In fact the complexity of the proteome reaches uncountable orders of magnitudes. This means that significant technical and data-analytic innovations will be needed for the full understanding of biology. Current st...
We investigated the molecular effects of glucosamine supplements, a popular and safe alternative to non-steroidal anti-inflammatory drugs, for decreasing pain, inflammation and maintaining healthy joints. Numerous studies have reported an array of molecular effects after glucosamine treatment. We questioned whether the differences in the effects ob...
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...
α-synuclein (aSyn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of aSyn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogen...
In a previous study, evidence was provided that indoor secondhand tobacco smoke (SHS) air pollution remains high in Lisbon restaurants where smoking is allowed, regardless of the protective measures used. The aim of this study was to determine in these locations the levels of polycyclic aromatic hydrocarbons (PAH) associated with the particulate ph...
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 pa...
Abstract In Saccharomyces cerevisiae, adaptation to hydrogen peroxide (H(2)O(2)) decreases plasma membrane permeability to H(2)O(2), changes its lipid composition and reorganizes ergosterol-rich microdomains by a still unknown mechanism. Here we show, by a quantitative analysis of the H(2)O(2)-induced adaptation effect on the S. cerevisiae plasma m...
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 eryth...
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. Ci...
Attempts to promote normal processing and function of F508del-CFTR, the most common mutant in cystic fibrosis (CF), have been described as potential therapeutic strategies in the management of this disease. Here we described the proteomic approaches, namely two-dimensional electrophoresis (2DE), mass spectrometry (MS), and bioinformatics tools used...
A comprehensive proteomic profiling of nasal epithelium (NE) is described. This study relies on simple subcellular fractionation used to obtain soluble- and membrane-enriched fractions followed by 2-dimensional liquid chromatography (2D-LC) separation and tandem mass spectrometry (MS/MS). The cells were collected using a brushing technique applied...
Complementary 2D-PAGE and 'shotgun' LC-MS/MS approaches were combined to identify medium and low-abundant proteins in sera of Cystic Fibrosis (CF) patients (mild or severe pulmonary disease) in comparison with healthy CF-carrier and non-CF carrier individuals aiming to gain deeper insights into the pathogenesis of this multifactorial genetic diseas...
The organization of respiratory chain complexes in supercomplexes has been shown in the mitochondria of several eukaryotes and in the cell membranes of some bacteria. These supercomplexes are suggested to be important for oxidative phosphorylation efficiency and to prevent the formation of reactive oxygen species. Here we describe, for the first ti...
The achievement and maintenance of a protein native conformation is a very complex cellular process involving a multitude of key factors whose contribution to a successful folding remains to be elucidated. On top of this, it is known that correct folding is crucial for proteins to play their normal role and, consequently, for the maintenance of cel...
F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defec...
Current clinical, laboratory or radiological parameters cannot accurately diagnose or predict disease outcomes in a range of autoimmune disorders. Biomarkers which can diagnose at an earlier time point, predict outcome or help guide therapeutic strategies in autoimmune diseases could improve clinical management of this broad group of debilitating d...
The aim of this work was to establish protein profiles in serum and nasal epithelial cells of cystic fibrosis individuals in comparison with controls, asthma and chronic obstructive pulmonary disease patients for specific biomarker signatures identification.
Protein extracts were analyzed by Surface Enhanced Laser Desorption/Ionization Time-Of-Flig...
To gain insight into the proteins potentially involved in the low temperature-induced F508del-CFTR rescue process, we have explored by two-dimensional electrophoresis (2DE) the proteome of BHK cell lines expressing wt or F508del-CFTR, grown at 37 degrees C or 26 degrees C/24h or 26 degrees C/48h followed by 3h of metabolic labelling with [(35)S]-me...
Combined results from laser capture microdissection of mouse airway epithelial cells followed by high power (MALDI-FTICR) MS, and fluorescent two-dimensional gel elctrophoresis (2D-DIGE) of the whole lung, allowed us to identify proteins differentially expressed after naphthalene induced airway injury. Further, we discovered several novel aspects o...
Hepatitis delta virus (HDV) infects human hepatocytes already infected with the hepatitis B virus increasing about ten fold the risk of cirrhosis and fulminant hepatitis. The lack of an appropriate cell culture system capable of supporting virus replication has so far impaired the detailed investigation of the HDV biology including the identificati...
The proteome project, initiated in 1995, was made possible by 2-DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2-DE/MS-based...
Plans for the European Proteomics Association (EuPA) were conceived and established during 2004 and 2005, and culminated in the formal inception of the organisation during the 4th HUPO World Congress held in Munich in 2005. The mission from the outset has been three-tiered and is to: i) strengthen the national Proteomics organizations in their effo...
Hepatitis delta virus (HDV) infection of human hepatocytes infected with the hepatitis B virus (HBV) is associated with increased liver damage and risk of fulminant disease. Although considerable progress has been made towards the elucidation of the mechanisms of HDV replication and pathogenesis, little is still known about the host factors involve...
Ceruloplasmin (CP) is a multicopper oxidase involved in the acute phase reaction to stress. Although the physiological role of CP is uncertain, its role in iron (Fe) homeostasis and protection against free radical-initiated cell injury has been widely documented. Previous studies showed the existence of two molecular isoforms of CP: secreted CP (sC...
The early transition of knowledge from highly specialised and sophisticated proteomics research to a diverse community in need of know-how is a challenge that requires backing from advanced research centres and groups, and a coordinating body for the dissemination of this knowledge. The European Proteomics Association (EuPA) Education Committee sig...
Genetically modified mouse strains are important research tools for the study of numerous human diseases. These models provide us with differentiated tissues, which are not often available from human sources. Furthermore, they allow for testing the effects of genetic manipulation and experimental therapeutics on physiology and pathology. Their impo...
Proteomics was initially viewed as a promising new scientific discipline to study complex disorders such as polygenic, infectious and environment-related diseases. However, the first attempts to understand a monogenic disease such as cystic fibrosis (CF) by proteomics-based approaches have proved quite rewarding. In CF, the impairment of a unique p...
A panel of 11 human cystic fibrosis transmembrane conductance regulator (hCFTR) antibodies were tested in ovine nasal, tracheal, and bronchial epithelial brushings. Two of these, G449 (polyclonal) and MATG1104 (monoclonal), recognized hCFTR but did not cross react with endogenous sheep CFTR. This specificity allows immunologic detection of hCFTR ex...
Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority...
The hepatitis delta virus (HDV) is the smallest human virus and the only satellite virus known so far. For the production of infectious particles the HDV requires the presence of the hepatitis B virus (HBV) surface proteins. Hepatitis delta patients develop more severe symptoms than patients infected only by the HBV. Due to its simplicity this viru...
F508del is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsible for the genetic disease Cystic Fibrosis (CF). It results in a major failure of CFTR to traffic to the apical membrane of epithelial cells, where it should function as a chloride (Cl-) channel. Most studies on localization,...
Tall columnar epithelial (TCE) cells can be obtained by a non-invasive procedure through brushing the inferior turbinate and the adjacent lateral nasal wall. Here, we present results from the functional study of epithelial cells, thus obtained by using the patch-clamp technique. By patch-clamping the sub-apical region of TCE cells, we were able to...
Studies on CFTR protein expression and localization in native tissues or in primary cultures of human epithelial cells are scarce due to the intrinsic instability of this protein, its low expression in most tissues and also to technical difficulties. However, such data are of the highest importance to understand the pathophysiology of CF. The purpo...
Reliable methods for determining the localisation of mutant CFTR protein in native cells from CF individuals are necessary to allow the degree of mislocalisation of any genotype to be defined and to assess the effect of therapeutic agents on CFTR trafficking. Here, we present procedures for obtaining ciliated epithelial cells from CF patients by na...
For most expression studies focusing on the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, sensitive and specific antibodies (Abs) are critically needed. Several Abs have been produced commercially or by research laboratories for CFTR detection in both cell lines with heterologous or endogenous expression and native cells/tissu...
Detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein is usually a difficult task to accomplish due to the low levels of expression and high turnover that this membrane protein is submitted to in the cell. Common biochemical methods can be used for the detection of CFTR but several critical points must be taken into accoun...
Numerous factors, other than mutations in the CFTR gene, affect the phenotypic variability of cystic fibrosis (CF). With a two-dimensional electrophoresis (2-DE) analysis of total protein expression profiles (proteomics) of CF versus non-CF cells it is possible to obtain an integrative picture of CF cellular alterations. Through this approach, prot...
Interest in the biochemical detection of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein followed soon after cloning of the gene and prediction of the protein structure. Ever since, antibodies (Abs) have been produced and used to detect CFTR in both heterologously and endogenously expressing cells and tissues. Although d...
The intracellular localization of cystic fibrosis transmembrane conductance regulator (CFTR) in native tissues is a major issue in the study of mutation, processing, and trafficking effects in CFTR and in the evaluation of therapeutic strategies in cystic fibrosis (CF). This work evaluated the applicability of ten different antibodies (Abs) under v...
A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Altogether, results presented...
Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl(-)) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations...
Estimates of the level of transcripts from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene required to develop a CF phenotype range from 4-20% of normal. Due to the importance of obtaining reliable data on this issue for therapeutic strategies, we developed a novel polymerase chain reaction-based method to quantify CFTR tra...
The CFTR (cystic fibrosis transmembrane conductance regulator) gene, defective in cystic fibrosis, codes for a polytopic apical membrane protein functioning as a chloride channel. Wild-type (wt) CFTR matures inefficiently and CFTR with a deletion of Phe-508 (F508del), the most frequent mutation, is substantially retained as a core-glycosylated inte...
Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization...
We characterized the 3272-26A-->G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26...
We characterized the 3272-26A→G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A→...
A Tetrahymena pyriformis gene orthologue to the mouse CCT gamma gene has been cloned and sequenced. The coding region of this gene, named TpCCT gamma, is interrupted by six introns and codes for a protein of 559 amino acid residues. This protein shares a 57.6% identity with the mouse CCT gamma protein whereas in average a 30% identity was found wit...
A Tetrahymena pyriformis gene orthologue to the mouse CCT gamma gene has been cloned and sequenced. The coding region of this gene, named TpCCT gamma, is interrupted by six introns and codes for a protein of 559 amino acid residues. This protein shares a 57.6% identity with the mouse CCT gamma protein whereas in average a 30% identity was found wit...
Projects
Projects (3)
Thousands of circulating proteins have been shown to be hallmarks of emerging disease, response to treatment, or a patients’ prognosis. The identification of these small molecule biomarkers holds a great promise for significant improvement of personalized medicine based on simple blood tests. For instance, diagnosis and prognosis with biomarkers (e.g. carcinoembryonic antigen (CEA)) has significantly improved patient survival and decreased healthcare costs in colorectal cancer patients. Unfortunately, despite significant investments to increase the number of biomarker studies, only ~150 out of thousands of identified biomarkers have currently been implemented in clinical practice. This is mainly caused by the time-consuming process of reliably detecting biomarkers, the irreproducibility of studies that determine a biomarkers’ clinical value, and by a mismatch in studies that are performed by academia and what is required for regulatory and market approval. To increase the number of clinically validated biomarkers, rather than further increasing the number of biomarker discovery studies, CliniMARK will improve the quality and reproducibility of studies and establish a coherent biomarker development pipeline from discovery to market introduction.
