Computational protocol: Trypanosomal mitochondrial intermediate peptidase does not behave as a classical mitochondrial processing peptidase

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[…] The gene encoding a putative MIP was identified using BLAST in the T. brucei genome database (www.tritrypdb.org). The T. brucei MIP sequence together with 32 selected MIP homologs, 7 genes of closely related thimet peptidases, 9 genes of OpdA and 6 genes of Dcp () obtained by a BLAST search were aligned using Muscle 3.8.425 software (default parameters) [] and trimmed with BMGE 1.12 (-b 5 -m BLOSUM30) []. Protein evolution model was selected by ProtTest 3.2 [] to reconstruct phylogenetic tree by PhyML 3.1 (topology search: best of NNIs and SPRs, initial tree: BioNJ, Substitution model: LG, proportion of invariable sites: fixed (0), gamma distribution parameter: estimated; number of categories: 4; bootstrap replicates: 500) [] and MrBayes 3.2.6 (rate matrix: LG; rate variation: gamma; gamma categories: 4; chain length: 2,000,000; heated chains: 4; heated chain temp: 0.2; burn-in length: 500,000) []. [...] Putative mitochondrial pre-sequences and the cleavage sites of MPP were predicted by Gavel’s consensus patterns search in the PSORTII program (http://psort.hgc.jp/form2.html) [,] and MitoProt II [], using the T. brucei mitoproteome dataset []. Proteins with predicted pre-sequences were further analyzed for the MIP cleavage motif (F/L/I)XX(T/S/G)XXXX [] within the octapeptide which immediately follows the MPP cleavage site. From the proteins found by this procedure, one was selected to be further analyzed for MPP and MIP proteolysis detection. […]

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