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

Abstract

The ubiquitin proteasome system (UPS) is responsible for the most of intracellular proteolysis in eukaryotes and the ubiquitination process occurs through the action 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. In parasitic 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 high identity to the mammals, being considered a target for treatment of leishmaniasis, however little is known about UPS in Leishmania genus. My PhD project intend to characterize the Leishmania infantum orthologous to the human genes SKP1, RBX1 and CUL1, which are components of the most studied E3 ligases in humans called CRL (Cullin-RING ligases). We showed through immunoprecipitation in mammalian cells the interaction of human CRLs components and the L. infantum orthologs, indicating that motifs responsible for the interaction are conserved, suggesting the presence of CRL complex in L. infantum. Promastigote parasites were 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 used to develop immunoprecipitation with anti-myc beads. The eluates were analyzed by mass spectrometry 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 is functionally 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 throughput gene analysis in parasites, and proposed to individually knockout and labeled each of these genes with a barcode nucleotide sequence in L. infantum. The null viable mutants will be pooled into a culture and assays of promastigotes proliferation, amastigotes differentiation and L. infantum cell cycle and morphology will be performed. Non viable mutants will be selected to be studied through conditional knockout. SKP1, CUL1 and their protein partners will be evaluated through cellular co-localization in the super-resolution microscope Elyra 7. Thus, this opportunity 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. (AU)