Browsing by Author "Sheehan, David"
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- Comparison of thiol subproteome of the vent mussel Bathymodiolus azoricus from different Mid-Atlantic Ridge vent sitesPublication . Company, Rui; Torreblanca, Amparo; Cajaraville, Miren; Bebianno, Maria João; Sheehan, DavidDeep-sea hydrothermal mussels Bathymodiolus azoricus live in the mixing zone where hydrothermal fluid mixes with bottom seawater, creating large gradients in the environmental conditions and are one of the most studied hydrothermal species as a model of adaptation to extreme conditions. Thiol proteins, i.e. proteins containing a thiol or sulfhydryl group (SH) play major roles in intracellular stress defense against reactive oxygen species (ROS) and are especially susceptible to oxidation. However, they are not particularly abundant, representing a small percentage of proteins in the total proteome and therefore are difficult to study by proteomic approaches. Activated thiol sepharose (ATS) was used for the rapid and quantitative selection of proteins comprising thiol- or disulfide-containing subproteomes. This study aims to isolate thiol-containing proteins from the gills of B. azoricus collected in distinct hydrothermal vents and to study the thiol-containing subproteome as a function of site-specific susceptibility to ROS. Results show that ATS is a powerful tool to isolate the thiol-containing sub-proteome and differently-expressed protein spots showed significant differences among the three vent sites, supporting previous findings that specific environmental conditions are crucial for ROS formation and that B. azoricus have different susceptibilities to oxidative stress depending on the vent site they inhabit.
- Ubiquitination and carbonylation as markers of oxidative-stress in Ruditapes decussatusPublication . Chora, Suze; McDonagh, Brian; Sheehan, David; Starita-Geribaldi, Mireille; Roméo, Michèle; Bebianno, Maria JoãoEnvironmental pollutants, such as metals, are widespread in aquatic environments and can lead to the formation of reactive oxygen species (ROS). ROS are highly toxic in marine species since they can cause serious reversible and irreversible changes in proteins including ubiquitination and modifications such as carbonylation. This study aimed to confirm the potential of ubiquitination and carbonylation as markers of oxidative stress in the clam Ruditapes decussatus (Veneroida, Veneridae) exposed to cadmium (40 microg/L). After 21 days of exposure clams were dissected into gills and digestive gland. Cytosolic proteins of both tissues were separated by two-dimensional electrophoresis (2-D SDS-PAGE) and analysed by immunobloting. Higher ubiquitination and carbonylation levels were in digestive gland of contaminated organisms. These results confirm the potential of ubiquitination and carbonylation as a sensitive and specific marker of oxidative stress in marine bivalves. In this approach, changes in protein structure provide options for affinity selection of sub-proteomes for 2D SDS-PAGE, simplifying the detection of protein biomarkers using proteomic approach.
- Ubiquitination and carbonylation of proteins in the clam Ruditapes decussatus, exposed to nonylphenol using redox proteomicsPublication . Chora, Suze; McDonagh, Brian; Sheehan, David; Starita-Geribaldi, Mireille; Roméo, Michèle; Bebianno, Maria JoãoUbiquitination and carbonylation of proteins were investigated in the gill and digestive gland of Ruditapes decussatus exposed to NP (nonylphenol) (100 μgL(-1)) using redox proteomics. After 21 d of exposure, clams were dissected and cytosolic proteins of both tissues separated by 2DE SDS-PAGE. Protein expression profiles were tissue-dependent and differently affected by NP exposure. Ubiquitination and carbonylation were also tissue-specific. NP exposure induced significantly more ubiquitinated proteins in gills than in digestive glands, compared to controls. Digestive gland showed a significant higher number of carbonylated proteins than gills after NP exposure. Protein ubiquitination and carbonylation are therefore independent processes. Results showed that NP exposure generated ROS in gill and digestive gland of R. decussatus that significantly altered the proteome. Results also highlighted the advantage of using redox proteomics in the assessment of protein ubiquitination and carbonylation, which may be markers of oxidative stress in R. decussatus.