Synthesis and incorporation of selenocysteines in Trypanosoma and Amoebae: structure, molecular interactions and relevance to the parasite.

• Grant number: 24/18708-4
• Support Opportunities: Research Projects - Thematic Grants
• Start date: November 01, 2025
• End date: October 31, 2030
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Otavio Henrique Thiemann
• Grantee: Otavio Henrique Thiemann
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Pesquisadores principais: João Renato Carvalho Muniz
• Associated researchers: Ana Joaquina Pérez Berná ; Andrea Sorrentino ; Ariel Mariano Silber ; Carla Cristina Polo ; Jonatas Santos Abrahao ; Julia Pinheiro Chagas da Cunha ; Juliana Reis Cortines ; Marcio Silva ; Vitor Hugo Balasco Serrão

Abstract

Protein translation processes play a central role in cellular metabolism and are present in all domains of life. They are therefore ideal markers for investigating the evolution of ancient eukaryotes. They also depend on extensive and specific protein-protein and protein-RNA interactions. In 1986, the 21st amino acid, selenocysteine (Sec - U), was discovered. Its incorporation into the UGA termination codon is directed by the structure in messenger RNA, the SECIS element, and a complex molecular machinery that our group has described in Escherichia coli, Trypanosoma and Naegleria. We recently identified the Sec synthesis pathway in Acanthamoeba and the presence of SECIS elements in the genomes of Tupanvirus and Niemeyervirus. These viruses belong to the Nucleocytoviricota phylum and replicate in Viral Factories (VF) in the cytoplasm of Acanthamoeba castellanii, encoding more than 1,000 genes, many of which are involved in translation processes. This project aims to further characterize this enzymatic pathway and aminoacyl-tRNA synthetases, describing their role in the physiology of protozoan parasites as well as in the replication cycle of Tupanvirus and Niemeyervirus. To this end, we propose: a- To determine the kinetic and thermodynamic parameters of protein-protein and protein-RNA interactions of the complexes formed in the Sec synthesis and incorporation pathway of T. cruzi, N. gruberi and A. castellanii. b- To characterize the structure of the complexes by small-angle X-ray diffraction, single-crystal X-ray diffraction and single-particle cryo-electron microscopy (Cryo-TEM). c- To develop knockout T. cruzi strains in order to investigate their impacts on the physiology of the parasite and thus determine possible therapeutic targets. d- To characterize the structure of Tupanvirus and Niemeyervirus and the VF of these viruses by Cryo-TEM, cryo-electron tomography (Cryo-ET) as well as by different biophysical techniques and mass spectrometry. e- To characterize biochemically and structurally the aminoacyl-tRNA synthetases of Niemeyervirus. Thus, the present proposal aims to describe the Sec synthesis pathway and the mechanisms of interaction and specificity between proteins and protein-RNA in basal organisms of the eukaryotic phylogenetic tree, characterize its potential as a target in the design of inhibitors as well as characterize the replication cycle of Nucleocytoviricota (Tupanvirus and Niemeyervirus) in A. castellanii and its relationship with selenoproteins. (AU)