Biochemical characterization of F-box-like proteins (FLPs) in Leishmania infantum.

Abstract

Intracellular proteolysis in eukaryotes is primarily mediated by the Ubiquitin Proteasome System (UPS), which operates through the cascading action of three enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligases). These enzymes are essential in this process as they recognize and transfer ubiquitin to target substrates. Intracellular proteolysis plays a crucial role in parasitic protozoa, as it is important for host alternation in their life cycles and, consequently, for the success of parasitism. Nevertheless, the UPS remains poorly studied in most parasites, including the trypanosomatids of the Leishmania genus, responsible for causing various forms of neglected tropical diseases.Leishmania infantum, in particular, is the etiological agent of Visceral Leishmaniasis (VL) in Brazil, causing the most severe form of the disease, which can be fatal without proper treatment. The subunits of the Leishmania proteasome exhibit a high degree of identity to those of humans, making them potential drug targets for leishmaniasis treatment. Recent studies characterizing the E2 enzymes and deubiquitinases of L. mexicana have demonstrated their essential roles in proliferation, infection, and parasitic success. However, the E3 ubiquitin ligases of Leishmania remain poorly understood. In this project, we propose to biochemically characterize the genes LINF_140018100 (F-box like protein 3 - FLP3), LINF_330019800 (F-box like protein 4 - FLP4), LINF_340019100 (F-box like protein 5 - FLP5), and LINF_360032300 (F-box like protein 6 - FLP6) of L. infantum. These genes were identified as binding partners of SKP1 through mass spectrometry results obtained by our group (A.R. FAPESP 2020/15771-6) compiled in a forthcoming article. To achieve this, L. infantum parasites will be genetically modified using the CRISPR-Cas9 strategy to insert 3xmyc-mCherry tags into these genes and to create knockout strains. We will perform the biochemical characterization of these strains by identifying the ligands of the proteins encoded by these genes through immunoprecipitation of myc-tag-fused proteins, followed by mass spectrometry analysis. In the interactome, we will search for potential substrates of FLP3, FLP4, FLP5, and FLP6 of L. infantum to elucidate their functions in L. infantum. Viable knockout strains will be characterized in terms of growth rate and in vitro infection. Thus, the results of this project will contribute to our understanding of this parasite's physiology and may lead to the identification of new targets for pharmacological intervention aimed at leishmaniasis treatment.