Computational protocol: Molecular Analysis of Aedes aegypti Classical Protein Tyrosine Phosphatases Uncovers an Ortholog of Mammalian PTP 1B Implicated in the Control of Egg Production in Mosquitoes

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Protocol publication

[…] Identification and classification of tyrosine phosphatases (PTPs) in mosquitoes were accomplished by searching conserved domains from Pfam. The search for conserved domains (CDs) employed FAT , a program developed by our group. It is a HMMER filter and blast manager. First, proteins predicted (version 1.3) by Vector-Base in the A. aegypti genome were downloaded (https://www.vectorbase.org/download/aedes-aegypti-liverpoolpeptidesaaegl13fagz). Using FAT, the sequences were filtered using characteristic domains of PTP families (Class I soluble classical PTPs used Y_phosphatase domain PF00102). The sequences obtained were compared using blast with databases such as nr, Swiss-prot and a custom human tyrosine phosphatases database. Lastly, hmmscan searched for other conserved domains in these previously filtered proteins. While this manuscript was in preparation, we became aware of another publication that described a specific sequence-based method for the automatic classification of PTPs present in 65 genomes, including A. aegypti . The same classical PTPs described in that study were confirmed by the strategy described here. [...] In silico approaches were used to ascertain the function and the evolutionary homologs of Aedes PTPs. Because proteins with high sequence and structural similarity may possess the same biological functions, the in silico approach was used to find the closest homologs of PTPs in an effort to obtain homology gene models . Sequences were used to search the homologue sequence space for related proteins using the NCBI BLAST server . A BLOSUM62 matrix was used for coring of the sequences, with an expected threshold of 10 and a sampling size of 3 amino-acids at a time. The non-redundant protein database was used for searching the sequence space without filtering out low-complexity regions. To identify the homologues with known protein structures, the PSI-BLAST tool was used against the freely available Protein Data Bank. PSI-BLAST allowed for better sensitivity and enabled us to find the closest five structures, which could be used in homology modeling. These five sequences were used for a multiple sequence alignment using the MULTALIGN server –. In several instances, the Aedes PTP and the homologous PTP sequences had gaps, so the modeler could not be effectively used for homology modeling. In these cases, the automated Protein Homology/analogy Recognition Engine was used to find the respective homology models . The reliability of the models was checked via submission to the WHAT IF server , . The protein structures were visualized and superimposed using PyMOL software (DeLano Scientific LLC). The various domains in the sequences were identified using a combination of online servers, including the Conserved residue Function Prediction Server and the Conserved domain Database at the NCBI . The active site of the Protein-Tyrosine-Phosphatase domain was ascertained using the Catalytic Site Atlas as well as the siteFinder web-based tool . The ten motifs that define the PTP domain were identified and used, as defined in the PTP database . Secondary-structure prediction for the sequence corresponding to the PTP domain was obtained from the Psipred server . Sequences of human and fly (Drosophila melanogaster) PTPs were obtained from the non-redundant Protein Database at NCBI and used for construction of dendrograms using the CLUSTALW web server and the TREEVIEW software package. […]

Pipeline specifications

Software tools HMMER, HMMER hmmscan, BLASTN, BLASTP, WHAT IF, PyMOL, PSIPRED, Clustal W
Databases Pfam UniProt VectorBase CDD
Applications Drug design, Amino acid sequence alignment
Organisms Aedes aegypti, Homo sapiens
Diseases Diabetes Mellitus, Neoplasms, Obesity