Identification and purification of Leishmania (L.) amazonensis telomeric proteins

Author(s)

Cristina Braga de Brito Lira

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2007-06-22

Examining board members

Maria Isabel Nogueira Cano; Maria Julia Manso Alves; Fábio Cesar Gozzo; Jörg Kobarg; José Andrés Yunes

• Advisor: Maria Isabel Nogueira Cano

Abstract

Leishmaniasis is a parasitic disease that was classified by World Health Organization as a Category 1 disease because there are no effective control programs or therapeutics to this disease. Parasites from the Leishmania genus are the ethiologic agents of leishmaniasis and they possess linear chromosomes with telomeric ends. Telomeres are nucleoprotein specialized nucleoprotein complexes essential for maintaining chromosomal stability and cell viability. Since telomeric proteins are essential for the maintenance of telomeres, they could be considered good targets for anticancer and antiparasitic drugs. Therefore, the goal of this work was to identify Leishmania amazonensis proteins that interact with the telomeric DNA. Three methodologies were used the achive this goal: (i) purification of telomeric proteins from nuclear extracts of L. amazonensis, (ii) Leishmania genome database mining for protein containing a Myb-like domain, and (iii) use of One-hybrid system (Clontech). The search for telomeric proteins in nuclear extracts of L. amazonensis resulted in the identification of LaRbp38. EMSA and immunoprecipitation assays were used to attest LaRbp38 binding to telomeric, GT-rich and kinetoplast DNAs, both in vitro and in vivo. LaRbp38 could be considered a good drug target for antiparasitic therapy since it is exclusive of trypanosomatids and itshomologue in T. brucei is essential for parasite survival. In order to characterize structurally this protein, LaRbp38 was expressed in bacteria. The protein was present in the insoluble fraction of the bacterial lysate. Therefore, preliminary experiments of refolding were done. LaRbp38 seems to need DNA, or analog molecules, in order to correctly refold in vitro. Data-mining in the L. major genome resulted on a list of proteins bearing a Myb-like domain. One protein was chosen and its L. amazonensis homologue was termed LaTBP1. LaTBP1 Myb-like domain is centrally localized and shares sequence similarities with Myb-like domains found on transcription factors TFIIIB and c-MYB, and with the RAP1 telomeric proteins. Competition and chromatin immunoprecipitation assays confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs. This binding specifities are also found on the telomeric proteins Rap1, Taz1 and TEBP1, described in yeast and higher eukaryotes. A list of proteins with a putative telomeric function was generated after the use of the Onehybrid system. Sequence analysis (search for homologues in other organisms) and EMSA, done with protein extract of recombinant yeast, were used to infer a telomeric function for the proteins found by this methodology. Although the results are encouraging, detailed analysis are necessary to validate the interactions. In conclusion, the three methodologies were usefull for the identification of telomeric proteins. This work resulted in the identification of several telomeric candidate proteins (AU)