Characterization of the lysine acetylatransferase NAT10 from Leishmania mexicana using Saccharomyces cerevisiae as a model

• Grant number: 21/13714-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2022
• End date: December 31, 2022
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Nilmar Silvio Moretti
• Grantee: Gabriela Gomes Alves
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

Leishmania spp. it is the etiological agent of leishmaniasis, a group of diseases that can manifest in three main forms: cutaneous, mucocutaneous and visceral. The diversity of clinical cases presented varies according to the species of Leishmania, with more than 20 species that can affect humans being described. During its life cycle Leishmania spp. circulates between an invertebrate and a vertebrate host, and to survive the environmental alterations found it needs to adapt, which could involve changes in the gene expression, translation and metabolism. The regulation of gene expression in Leishmania occurs mainly at post-transcriptionally level, through mRNA stabilization mechanisms and translation regulation. Different post-transcriptional modifications have already been described for mRNAs, such as methylation and pseudouridylation, named epitranscriptome. It was recently described that mRNAs can be modified by acetylation in mammalian cells, a modification previously described only for tRNAs and rRNAs, by the activity of N-acetyltransferase, NAT10. The acetylation of mRNAs occurs in cytidines and is called N4-acetylcytidine (ac4C), having a great impact on promoting an increase in the stability of mRNAs and promoting an increase in the efficiency of translation. Considering that post- transcriptional regulation is crucial for Leishmania spp., in this project we will study the role of mRNA acetylation in this parasite. Using bioinformatics analysis we identified the homologous gene of NAT10 in Leishmania mexicana, and we demonstrated the presence of ac4C in different species of the parasite. Thus, to better understand the role of this protein, we intend in this project to evaluate the functional complementation of the NAT10 gene in the Saccharomyces cerevisiae model. For this, we will: I) Obtain knockout strains of S. cerevisiae for the NAT10 gene; II) Cloning the L. mexicana gene into S. cerevisiae expression vectors; III) Obtain knockout strains of S. cerevisiae complemented with NAT10 gene from L. mexicana; IV) Evaluate the complementation of the S. cerevisiae NAT10 function by the L. mexicana protein through diferente phenotypic assays; V) Investigate the levels of ac4C in the S. cerevisiae cell lines generated. We hope with this project to contribute to a better understanding of an essential mechanism for Leishmania and to complement other ongoing projects in our group related to the characterization of NAT10 in L. mexicana.(AU)