Multiple types of nuclear localization signals in Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite that belongs to the Amoebozoa supergroup whose study related to the nucleocytoplasmic transport of proteins through the nucleus is poorly studied. In this work, we have performed in silico predictions of the potential nuclear localization signals (NLS) corresponding to the proteome of 8201 proteins from Entamoeba histolytica annotated in the AmoebaDB database. We have found the presence of monopartite nuclear localization signals (MNLSs), bipartite nuclear localization signals (BNLSs), and non-canonical monopartite NLSs with lengths exceeding 20 amino acid residues. Additionally, we detected a new type of NLS consisting of multiple juxtaposed bipartite NLSs (JNLSs) that have not been described in any eukaryotic organism. Also, we have generated consensus sequences for the nuclear import of proteins with the NLSs obtained. Docking experiments between EhImportin α and an MNLS, BNLS, and JNLS outlined the interacting residues between the Importin and cargo proteins, emphasizing their putative roles in nuclear import. By transfecting HA-tagged protein constructs, we assessed the nuclear localization of MNLS (U1A and U2AF1), JMNLS (U2AF2), and non-canonical NLS (N-terminus of Pol ll) in vivo. Our data provide the basis for understanding the nuclear transport process in E. histolytica.