Biochemical and functional characterization of NAT10 enzyme from Leishmania

Abstract

Leishmania spp. is the etiological agent of leishmaniasis, a group of diseases that can manifest in three main forms: cutaneous, mucocutaneous and visceral. During its life cycle Leishmania spp. circulates between two hosts, one invertebrate and one vertebrate, and during the transition between both it needs to adapt to the environmental changes found, and for that, the parasite uses changes in gene expression, translation and metabolism to survive. The regulation of gene expression in Leishmania occurs post-transcriptionally, through mRNA stabilization mechanisms and protein synthesis regulation. Several post-transcriptional modifications have already been described for mRNAs, such as methylation and pseudouridylation, which together are called epitranscriptome. Recently was verified that mRNAs can also be modified by acetylation, a modification previously described only for tRNAs and rRNAs, by the activity of the N-acetyltransferase enzyme, NAT10. The acetylation of mRNAs occurs in cytidines and is called N4-acetylcytidine (ac4C), and depending on the region of the mRNA that is present, it can promote an increase/decrease in the stability of the mRNAs and/or changes the efficiency of the mRNA translation. Whereas post-transcriptional regulation is important for Leishmania sp. and the role that ac4C may have in regulating the lifespan of mRNAs, in this project we will study the role of mRNA acetylation in this parasite, through the biochemical and functional characterization of the NAT10 protein in Leishmania mexicana. For that, we will use different approaches of molecular biology and biochemistry to obtain the heterologous protein NAT10 from L. mexicana for activity assays and we will functionally validate the function of NAT10 using both Saccharomyces cerevisiae and L. mexicana models. In the end, with this project, we aim to contribute to a better understanding of the regulatory mechanisms of Leishmania gene expression and the role of ac4C in mRNAs.