Computational protocol: Emergence, development and diversification of the TGF-β signalling pathway within the animal kingdom

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Protocol publication

[…] Known human Smads and TGF-β receptor proteins were used for a BLASTP search against a collection of proteomes predicted for high-coverage sequenced genomes, providing as wide as possible coverage of the animal kingdom. BLASTP parameters were first calibrated to yield searches of optimal sensitivity and specificity using human and mouse genomes (where the identity of relevant genes is well known) and verified using more distantly related animal genomes, through manual inspection of hits and alignments (to avoid, for example, non-specific hits to the kinase domain of the receptors). The following E-value cut-offs were used: 10e-30 for receptors and 10e-20 for Smads.It is important to notice that searches against the collection of proteomes were unbiased by the identity of species used as the starting point. No additional genes can be identified when searching with D. melanogaster, Nematostella, Trichoplax or Lottia gigantea Smads and receptors. In fact, these proteins are so well conserved in sequence that searches starting with genes originating from different phyla are essentially equivalent. For example, when Smads and receptors from human, D. melanogaster, Nematostella, Trichoplax or Lottia gigantea were used as queries against their proteomes as well as those of Xenopus tropicalis, Monodelphis domestica, Danio rerio, Ciona savignyi, and Caenorhabditis elegans, identical lists of hits were obtained (except that query using Trichoplax receptors did not detect one gene in human, M. domestica and X. tropicalis).The lists of homologs were further filtered, in order to include only those proteins which contained an exemplary Pfam domain []: MH2 for Smads; and any of the following for TGF-β receptors: an activin-type I/II receptor domain, a TGF-β receptor domain, or a TGF-β-GS motif for type I receptors [see Additional file ]. Presence of the terminal phosphorylation motif (SSxS) was also verified in case of R-Smads. Multiple sequence alignments were performed using Muscle []. […]

Pipeline specifications

Software tools BLASTP, MUSCLE
Databases Pfam
Application Nucleotide sequence alignment
Organisms Trichoplax adhaerens, Homo sapiens, Caenorhabditis elegans