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Testing the interactions between the telomeric proteins LaRBP38 and LaRPA-1 using the yeast two-hybrid system

Grant number: 11/22879-9
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): February 01, 2012
Effective date (End): December 31, 2012
Field of knowledge:Biological Sciences - Genetics
Principal Investigator:Maria Isabel Nogueira Cano
Grantee:Douglas Diez Gonçalves
Home Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Testing the interactions between the telomeric proteins LaRbp38 and LaRPA-1 using the yeast two-hybrid systemLeishmaniasis is a group of infectious diseases caused by parasites of the genus Leishmania, which affect millions of people around the world, with two million of new cases and 70,000 deaths each year. Drugs used to treat different clinical forms of the disease have high toxicity and low efficacy, in addition to cause many side effects. Moreover, the emergence of resistant parasites and vectors argue in favor of the development of new therapies to eradicate the disease, which is largely encouraged by the World Health Organization. Thus, a better understanding of the molecular biology of these protozoa may facilitate these discoveries. Actually, telomeres, the nucleoprotein structures at the ends of eukaryotic chromosomes, have been the subject of intensive studies, since they are been considered potential therapeutic target against malignant tumors and pathogenic microorganisms. Telomeres typically appear as dynamic structures where interactions between DNA and proteins result in the formation of a high order telomeric complex, responsible for the protection and maintenance of chromosomes and genome stability and subsequently cell viability. Components of Leishmania amazonensis telomeric complex were identified using different biochemical approaches. Among them were identified LaRPA-1 (L. amazonensis Replication Protein A-1) and LaRbp38 (L. amazonensis RNA binding protein) which showed the ability to interact with telomeric DNA in vitro and in vivo. More recently, we were able to show by immunoprecipitation and "pull-down" assays, that these proteins are part of common complex at parasite telomeres. This work aims to confirm these possible interactions, using an in vivo assay, known as the yeast two-hybrid system. One of our main objective is to standardize the method in our laboratory, since it will allow to confirm in a feasible manner, most of protein:protein interactions that we are testing in the lab. To facilitate the analysis of the results and unequivocally check the interactions and differentiate them from auto-activation and false positivies, both proteins (LaRPA-1 and LaRbp38) will be tested individually and as bait (fused to the DNA binding domain of LexA) and prey (fused to the GAL4 activation domain).