Obtaining a new recombinant protein component of the telomeric complex LAGT1 from Leishmania amazonensis

• Grant number: 12/17446-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2012
• End date: October 31, 2013
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Maria Isabel Nogueira Cano
• Grantee: Bárbara Moraes Souza
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Among the Leishmania genus are the causative agents of leishmaniasis, a medical important disease that presents a large spectrum of clinical presentations. The protozoa nuclear genome has 70Mb distributed in 34-36 chromosomes. Telomeres of L. amazonensis, one of the main etiologic agents of Tegumentar leishmaniasis in the Americas, is composed of the conserved repetitive sequence 5'-TTAGGG-3', which is maintained by telomerase. In this protozoa different proteins: telomeric DNA complexes were characterized and three complexes, named LaGT1-3, showed to associate more specifically in vitro and in vivo with the G-rich telomeric strand. LaRbp38 and LaRPA-1 were respectively identified as the components of LaGT2 and LaGT3 and the characterization of the ~ 15 kDa protein component of LaGT1 is the main goal of the present project. Previous results from our group showed that LaGT1 complex is very abundant and binds specifically the G-rich telomeric single-stranded DNA in the presence of high salt concentration or when subject to a variety of temperatures. Using mass spectrometry and de novo sequence analysis its protein component was identified as a putative protein sequence annotated as CALA2 or calmodulin. In L. major genome database, the gene has three copies organized in tandem (LmjF09.0910, LmjF09.0920 e LmjF09.0930). The identification of the protein as a calmodulin-like protein indicates that it shares with other related proteins that bind calcium to one or more structural calcium-binding domains (e.g. EF-hand domains). In this context, a DNA-PKc-interacting protein (KIP), which has an EF-hand domain and shares high homology with calcineurin and calmodulin, was described as a human telomerase component. Therefore, based in this information, the protein component of LaGT1 can be considered a new and exclusive parasite protein, which would be an important target for anti-parasite drug design. The present project has the aim of cloning the gene encoding the putative CALA2 from L. amazonensis, and obtaining the recombinant protein using the pQE2 bacterial expression system. The recombinant protein will be affinity purified and its secondary structure will be determined using circular dichroism. If we succeed, the recombinant protein will be used in protein: DNA interaction assays using the G-rich telomeric sequence as bait.(AU)