Study of the selenocysteine incorporation pathway: understanding the macromolecular interaction mechanism

Abstract

Naegleria fowleri, commonly known as "brain-eating amoeba", is the only species of Naegleria known to infect humans, resulting in the condition known as Primary Amebic Meningoencephalitis (PAM). Despite its medical relevance it is still an organism with little attention. Our group described the presence of the selenocystein synthesis pathway in N. gruberi, a nonpathogenic species used as a model system for Naegleria studies and has contributed to elcidade this pathway both in eukaryotes as well as in prokaryotes. Selenocysteine (Sec) is incorporated into nascent protein in a UGA codon by a co-translational event both in prokaryotes and in eukaryotes. In bacteria, the Sec synthesis pathway is composed of the enzymes Selenocysteine Synthase (SELA), a Specific Elongation Factor (SELB), Selenophosphate Synthetase (SELD), seryl-tRNA synthetase (SerRS) and a Selenocysteine lyase (CSDB). The Sec synthesis and incorporation route also depends on two RNAs; a specific tRNA (tRNASec) and a specific sequence in the mRNA (Selenocisteínas Insertion Sequence - SECIS), signaling to correct incorporation of Sec codon UGA. In eukaryotes, this pathway differs by the presence of O-fosfoseril-tRNASec Kinase (PSTK) and Selenocisteinil-tRNASec Synthase (SepSecS), replacing SelA, and the presence of SECIS binding proteins (SBP2) plus a factor Specific elongation factor (EFSec). This proposal aims at biochemical and structural studies of the proteins SELD CSDB of Escherichia coli and SBP2 of N. gruberi, to establish the molecular interactions that determine the specificity of these enzymes. Testing of interaction between CSDB-SelD and SBP2-SECIS will be conducted to determine the interaction constants through spectroscopic and calorimetric techniques. SBP2 in vivo immunofluorescence tests will be performed. Moreover, crystallization tests of different complexes and X-ray small angle scattering (SACS) will be performed to obtain structural models. Thus, this project aims to increase our knowledge and understanding of the molecular interactions present in selenocysteine synthesis route, being of relevance in the global understanding of the molecular interaction determinants between protein-protein and protein-RNA. (AU)