Functional characterization of proteins from the Ubiquitin Proteasome System of L. infantum

Abstract

This scientific initiation scholarship quota within the PI-FAPESP proposal aims at the production of gene knockout mutants labeled with barcodes of genes related to the ubiquitination process by Cullin1-RING ubiquitin ligases (CRL1) of L. infantum. The characterization of the effects of gene knockouts in viable null mutants will be performed through comparative assays of isolated culture with the wild type isolate of the parasite under the same experimental conditions. Among the analyses to be conducted, cellular growth will be highlighted by comparing their growth curves and effects on the cell cycle, performing flow cytometry. This class of E3 ligases, which is part of the PI project, is composed of F-box proteins, SKP1, Cullin1, and RBX1, and was recently described in this parasite by our group (2020/15771-6) in a paper submitted to the journal Plos Pathogens. The genes LINF_240015400, LINF_150014400, LINF_140018100, LINF_330019800, LINF_340019100, and LINF_360032300 encoding F-box-like proteins (Flps) 1 to 6 were found to interact with SKP1, indicating their functional relevance in the LinfCRL1 complex of L. infantum. In addition to these, the gene LINF_340026400 (Ubiquitin-like protein) was found in the interactome of Cullin1, potentially being the NEDD8 protein, a regulator of the assembly of the LinfCRL1 complex that covalently conjugates to Cul1 in active complexes. In this project, we will generate null mutants by CRISPR-Cas9 containing barcodes (Bar-seq), to be used within the PI project along with the other knockout clones that will be generated. The results of this project will contribute to the objectives of the PI grant, providing results on part of the genes of the Ubiquitin Proteasome System that will be studied. Together, this IC project will study 7 genes still unknown in L. infantum, which are part of a refined intracellular degradation system essential for the host alternation of this parasite. Thus, we will contribute to the understanding of a recently discovered system of E3 ligases that could be used as targets for the design of leishmanicidal drugs.