Functional characterization of the CRLs (Cullin RING ligases) E3 ubiquitin-ligases in Leishmania infantum

Abstract

The ubiquitin proteasome system (UPS) is responsible for the most of intracellular proteolysis in eukaryotes and the ubiquitination process occurs through the action of three enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-carrying enzyme) and E3 (ubiquitin-ligases) that play a key role in this process, recognizing and transferring ubiquitin to its substrate. These ubiquitinated targets can be directed to the proteasome for its degradation or have their functions regulated by this modification. In parasitic protozoan, intracellular proteolysis is essential for the alternation of hosts in their life cycles and consequently for the success of parasitism. Little is known about SUP in many parasites, including trypanosomatids of the Leishmania genus, responsible for causing leishmaniasis. Leishmania infantum is the etiologic agent of visceral leishmaniasis (VL) in Brazil, being the most severe form of the disease, which can progress to death if untreated. The Leishmania proteasome has a high identity to that of humans, being considered a target for treatment of leishmaniasis. Recently, the enzymes E2 and deubiquitinases from Leishmania mexicana were characterized, demonstrating their essential role in proliferation and infectivity. On the other hand, the E3 ubiquitin ligases of Leishmania remain unknown. Here, we propose to characterize the Leishmania infantum genes LINF_110018100, LINF_210005300 and LINF_240029100 that are orthologous to the human genes SKP1, RBX1 and CUL1 respectively, which are components of the most studied class of E3 ligases in humans called Cullin RING Ligases (CRLs) or SCF (SKP, Cullin, F-box). For this, we will genetically modify L. infantum through the CRISPR-Cas9 strategy, and produce knockout strains or expressing these genes in fusion with the 3xmyc-mCherry tags. We will perform the phenotypic characterization of the strains (growth, infectivity and susceptibility to drugs in vitro) and biochemistry studies, through immunoprecipitation of proteins in fusion with myc tag and analysis by mass spectrometry to identify the ligands. We will search in the interactome of L.infantum SKP, Cullin and RBX the potential components of the CRLs of this parasite and validation tests of protein-protein interaction and ubiquitination in vitro and in vivo will be performed. The results of this project will contribute to the knowledge of the physiology of this parasite and its relationship with the host, and may lead to the identification of new targets for pharmacological intervention aimed at the treatment of leishmaniasis. (AU)