Functional characterization of Leishmania infantum SKP1 and Cullin 1 interactome through CIRSPR-Cas9 bar-seq strategy

• Grant number: 23/07201-3
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: October 01, 2024
• End date: September 30, 2025
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Felipe Roberti Teixeira
• Grantee: Camila Rolemberg Santana Travaglini Berti de Correia
• Supervisor: Richard McCulloch
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Institution abroad: University of Glasgow, Scotland
• Associated to the scholarship: 21/10971-0 - Biochemical characterization of CRLs (Cullin RING ligases) E3 ubiquitin-ligases in Leishmania infantum, BP.DD

Abstract

The ubiquitin proteasome system (UPS) is responsible for the most of intracellular proteolysisin eukaryotes and the ubiquitination process occurs through the action of three enzymes: E1(ubiquitin-activating enzyme), E2 (ubiquitin-carrying enzyme) and E3 (ubiquitin-ligases) thatplay a key role in this process, recognizing and transferring ubiquitin to its substrate. Inparasitic protozoan, intracellular proteolysis is essential for the alternation of hosts in their lifecycles and consequently for the success of parasitism. The Leishmania proteasome has a highidentity to the mammals, being considered a target for treatment of leishmaniasis, however littleis known about UPS in Leishmania genus. My PhD project intend to characterize theLeishmania infantum orthologous to the human genes SKP1, RBX1 and CUL1, which arecomponents of the most studied E3 ligases in humans called CRL (Cullin-RING ligases). Weshowed through immunoprecipitation in mammalian cells the interaction of human CRLscomponents and the L.infantum orthologs, indicating that motifs responsible for the interactionare conserved, suggesting the presence of CRL complex in L.infantum. Promastigote parasiteswere genetically modified through CRISPR-Cas9 strategy to generate L. infantum lineages withSKP1 and Cullin1 in fusion with 3xmyc-mCherry and the extracts of these lineages were usedto develop immunoprecipitation with anti-myc beads. The eluates were analyzed by massspectrometry and SKP1 interactome reveal 43 unique proteins, including Cullin1, RBX1 and 6F-box domain containing proteins. Interestingly, Cullin1 interacting proteins included SKP1,RBX1 and one F-box protein among 22 protein partners. The aim of this BEPE proposal isfunctionally to characterize the SKP1 and CUL1 interactome identified in L.infantum. To that,we collaborated with Dr. Richard McCulloch, which group is specialized in high throughputgene analysis in parasites, and proposed to individually knockout and labeled each of thesegenes with a barcode nucleotide sequence in L. infantum. The null viable mutants will bepooled into a culture and assays of promastigotes proliferation, amastigotes differentiation andL. infantum cell cycle and morphology will be performed. Non viable mutants will be selectedto be studied through conditional knockout. SKP1, CUL1 and their protein partners will beevaluated through cellular co-localization in the super-resolution microscope Elyra 7. Thus, thisopportunity will be essential to ongoing PhD project and will give me expertise to apply high-throughput gene study and parasite characterization in our group in Brazil.