The discovery of new amino acids such as selenocysteine and pirrolisina, resulting in the expansion of the genetic code of the traditional 20 amino acids to currently 22 amino acids, have attracted the interest of several research groups. The synthesis pathway of selenocysteine (Sec - U) represents the major biological form of selenium, whose incorporation occurs co-translational form in selenoproteins and depends on a stop codon UGA in reading phase and a tertiary structure in messenger RNA known as element SECIS. Recently, we identified the existence of the route of synthesis of selenocysteine in kinetoplastid and characterized all genes of this pathway: SelB (Elongation Factor EFSec), SelD (Selenophosphate sintetase), PSTK (Fosfoseril tRNA kinase), SecSepS (Selenocysteine Sintase), SECp43 and tRNA[ser]sec. Furthermore, we identified three selenoproteins, SelK, SelT and SelTryp, and the last member has no identity with mammalian selenoproteins. However, details about composition and structure of the Sec incorporation complex are not known and remain obscure the role of SECIS elements in directing this process. So, the intention of project is identify proteins participant of Sec incorporation complex, using technique of complex purification such as PTP (ProtC - TEV - ProtA)-tag, have as bait trypanosome PSTK and SelB. As selenocysteine biosynthesis pathway evolution in primitive eukaryotes remains unclear, our group initiated the investigation of the selenocysteine incorporation machinery in Naegleria gruberi after its genome sequence was published. Interestingly, the SelD of N. gruberi has two distinct domains. The N-terminal has a predicted methiltransferase activity and the C-terminal is homologue to SPS/SelD. The aim of this part of project is study the probable participation of N-terminal domain in selenium detoxification. However, the identification of selenocysteine machinery in N. gruberi increase the informations into this pathway in primitive eukaryotes, little is known about which organisms are able to synthesis selenocysteine. Therefore, the last objective is search for selenocysteine incorporation pathway in other primitive organisms and increases the evolution information about this way in different eukaryotes using the next generation sequencing plataforms.
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