Functional characterization of Ubiquitin Proteasome System genes of Leishmania infantum

• Grant number: 24/17930-5
• Support Opportunities: Scholarships in Brazil - Master
• Start date: December 01, 2024
• End date: September 30, 2026
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Felipe Roberti Teixeira
• Grantee: Caroline Torres
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Associated research grant: 22/02933-3 - Role of the ubiquitin proteasome system in proliferation, differentiation and infectivity of Leishmania infantum, AP.PNGP.PI

Abstract

The ubiquitin-proteasome system (UPS) is responsible for most of the intracellular proteolysis in eukaryotes and consists of three enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-carrying enzyme) and E3 (ubiquitin-ligase enzyme). These enzymes act in a coordinated manner in the ubiquitination process, recognizing target proteins and transferring ubiquitin to them, thus directing them for degradation by the proteasome. In protozoan parasites, intracellular proteolysis is essential for host alternation in their life cycles and consequently crucial for the success of parasitism. In parasites of the genus Leishmania, the UPS is essential for their life cycle and proliferation and is a pharmacological target for the treatment of leishmaniasis, a group of neglected diseases that affect millions of people worldwide. However, despite the relevance of SUP for Leishmania, studies characterizing E3 ubiquitin ligases in these parasites are scarce. In this project, we propose to biochemically characterize the L. infantum genes LINF_070009100, LINF_060005200, LINF_150022200, LINF_080017100 and LINF_280024600, which are orthologous to the human genes TRAF6, COP1, SYVN, BRCA1 and Trim32, respectively, and which are part of the class of E3 ligases in humans called single RING ligases. Therefore, L. infantum parasites will be genetically modified using the CRISPR-Cas9 strategy to produce knockout strains marked by a unique nucleotide sequence (barcode). The viable strains will be pooled in a single promastigote culture (pooled culture - PC) that will be pooled with other knockouts of other SUP proteins and cultured for different times (0, 24, 48 and 168h). Promastigotes will also be differentiated into amastigotes and macrophages will be infected. Using next-generation sequencing of PCR products that will amplify the barcode regions (Bar-seq), bioinformatics analyses will be performed to evaluate the genes required for parasite development in each situation. Also using the CRISPR-Cas9 strategy, 3xmyc tags will be inserted into these genes for the biochemical characterization of these strains. The ligands of the proteins encoded by these genes will be identified through immunoprecipitation of the proteins bound to the 3xmyc tag, followed by mass spectrometry analysis to identify the ligands. We will search for other potential components of the SUP of this parasite in the interactome of the proteins TRAF6, COP1, SYVN, BRCA1 and Trim32 of L. infantum. Finally, assays to validate protein-protein interactions and in vitro ubiquitination will be performed. This proposal will allow the characterization of E3 ligases of L. infantum that are orthologous to enzymes responsible for essential processes in H. sapiens, such as apoptosis, proliferation and cell division. The results will contribute to understanding the physiology of the parasite and its cellular mechanisms for parasite-host interaction, leading to the identification of potential targets for pharmacological intervention aiming at the discovery of an effective treatment for leishmaniasis.