Are Leishmania infantum genes orthologous to humans SKP, CUL1 and RBX capable of expressing and assembling CRL 1-type E3 ubiquitin-ligase complexes (Cullin 1 RING-ligase) in human cells?

Abstract

The ubiquitin proteasome system (SUP) is primarily responsible for intracellular proteolysis in eukaryotes, consisting of 3 enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-carrying enzyme) and E3s (ubiquitin-ligases), which recognize and transfer ubiquitin to the substrate, which may subsequently be destined for degradation via the proteasome. In mammals, the largest and best-studied class of E3 ligases are the multi-subunit-like CRLs (Cullin RING-ligases), which are formed by the union of proteins SKP1, Cullin1, RBX1 and an F-box-like protein. In parasitic protozoa, intracellular proteolysis is fundamental for the alternation of hosts in their life cycles and consequent success of parasitism. In parasites of the genus Leishmania, which cause leishmaniasis, a group of neglected infectious diseases that affect millions of people, SUP has fundamental relevance to ensure cellular homeostasis and proliferation of these parasites, and is currently considered a pharmacological target for the treatment of leishmaniasis. However, despite the importance of SUP in Leishmania spp., there are no characterization studies of CRL-type E3 ubiquitin-ligase enzymes (Cullin RING-ligases) in parasites of this genus. Thus, our research group seeks to biochemically characterize the genes of L. infantum LINF_110018100, LINF_210005300 and LINF_240029100 which are orthologous to the human genes SKP1, RBX1 and CUL1 respectively. In silico analyses, we observed that these Leishmania spp. present a high degree of identity with their human orthologs, especially in interaction regions, suggesting a possibility of the existence of these complexes in the parasite. Therefore, we propose this project with the aim of evaluating whether the parasite's genes are expressed and capable of assembling CRL complexes in human cells. For this, we will clone the genes of L. infantum LINF_110018100, LINF_210005300 and LINF_240029100 in pcDNA3.1(+) vectors to generate clones pcDNA3.1(+)-LINF-SKP-FLAG, pcDNA3.1(+)-LINF-Cullin 1-myc and pcDNA3.1(+)-LINF-RBX-HA. Then, we will use mammalian cells (HEK293T) transfected with these vectors and perform immunoprecipitation with anti-FLAG-agarose and analyze the eluates by SDS-PAGE and western blotting. The results of this project may suggest the presence of CRLs in Leishmania spp. and will contribute to the knowledge of the physiology of this parasite and its relationship with the host, which may lead to the identification of new targets for pharmacological intervention aimed at the treatment of leishmaniasis.