RESUMEN
Because of their ecological importance, amphipod crustacea are employed worldwide as test species in environmental risk assessment. Although proteomics allows new insights into the molecular mechanisms related to the stress response, such investigations are rare for these organisms because of the lack of comprehensive protein sequence databases. Here, we propose a proteogenomic approach for identifying specific proteins of the freshwater amphipod Gammarus fossarum, a keystone species in European freshwater ecosystems. After deep RNA sequencing, we created a comprehensive ORF database. We identified and annotated the most relevant proteins detected through a shotgun tandem mass spectrometry analysis carried out on the proteomes from three major tissues involved in the organism's reproductive function: the male and female reproductive systems, and the cephalon, where different neuroendocrine glands are present. The 1,873 mass-spectrometry-certified proteins represent the largest crustacean proteomic resource to date, with 218 proteins being lineage specific. Comparative proteomics between the male and female reproductive systems indicated key proteins with strong sexual dimorphism. Protein expression profiles during spermatogenesis at seven different stages highlighted the major gammarid proteins involved in the different facets of reproduction.
Asunto(s)
Anfípodos/genética , Genoma , Proteoma/genética , Espermatogénesis/genética , Animales , Copulación/fisiología , Bases de Datos de Proteínas , Femenino , Agua Dulce , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Masculino , Anotación de Secuencia Molecular , Reproducción/genética , Caracteres SexualesRESUMEN
Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections.
Asunto(s)
Linfocitos T CD4-Positivos/química , Epítopos de Linfocito T/química , Genoma Viral , Antígenos HLA-DR/química , Virus Vaccinia/genética , Virus de la Viruela/genética , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Simulación por Computador , Epítopos , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Humanos , Datos de Secuencia Molecular , Unión Proteica , Virus Vaccinia/inmunología , Virus de la Viruela/inmunologíaRESUMEN
SUMMARY: Improving and ascertaining the quality of a multiple sequence alignment is a very challenging step in protein sequence analysis. This is particularly the case when dealing with sequences in the 'twilight zone', i.e. sharing < 30% identity. Here we describe INTERALIGN, a dedicated user-friendly alignment editor including a view of secondary structures and a synchronized display of carbon alpha traces of corresponding protein structures. Profile alignment, using CLUSTALW, is implemented to improve the alignment of a sequence of unknown structure with the visually optimized structural alignment as compared with a standard multiple sequence alignment. Tree-based ordering further helps in identifying the structure closest to a given sequence.