Functional and structural characterization of KKT2 and KKT9 kinetochore proteins of Trypanosoma Cruzi

Abstract

Cell division and the correct transmission of the genetic material are essential to the survival of living beings. A variety of proteins ensures the accuracy and timing of these events thus modifications or changes may result in extensive damage to the organism or death. Characteristics of the DNA replication machinery are shared among many eukaryotes meanwhile the molecules involved in the chromosome segregation have greater divergence.The identification of centromeric components (DNA and proteins) as well as kinetoplastids kinetochore proteins was difficult due the divergence among the primary sequence of other eukaryotes and Trypanosoma cruzi sequences deposited in the database. New approaches were needed to achieve these goals. Recently, 19 kinetochore proteins of Trypanosoma brucei were determined by Mass Spectrometry after isolation of protein complexes associated to proteins whose transcripts were increased during the cell cycle. The presence of KKT proteins (kinetoplastid kinetochore protein) in T. cruzi suggests these proteins may have the same role in the cell division.This project aims the in silico analysis of T. cruzi KKT orthologues, as well as the structural and functional characterization of two proteins, named KKT2 and KKT9. Considering the importance of the kinetochore in chromosome segregation and the fact that KKT are kinetoplastid-specific proteins, this study can result in potential targets for the development of new drugs with no side effects. The KKT2 and KKT9 genes will be cloned into expression vectors in bacteria in order to generate tools (recombinant protein, specific antibodies) which can help us to elucidate the role of these proteins. The expression of native KKT2 and KKT9 proteins during the T. cruzi cell cycle will be analyzed by western blot and confocal microscopy using polyclonal antibodies.