Biochemical characterization of 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, which is composed 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, which can be directed to the proteasome for its degradation. In parasitic protozoan, intracellular proteolysis is essential for the alternation of hosts in their life cycles and consequently for the success of parasitism. In Leishmania SUP is essential for the cell cycle and proliferation, being considered a target for treatment of Leishmaniasis, group of neglected diseases that affect millions of worldwide people. However, despite the relevance of SUP for Leishmania, there are no studies characterizing E3 ubiquitin-ligases in these parasites. Here, we propose biochemical characterization of 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). For this, we will genetically modify L. infantum through the CRISPR-Cas9 strategy, and produce strains expressing these genes in fusion with the 3xmyc-mCherry tags. We will perform 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 SKP, Cullin and RBX interactome of L.infantum the potential components of the CRLs in 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 the treatment of Leishmaniasis. (AU)