Caracterização in silico do Complexo E3 Ubiquitina-ligase LinfCrl1 (Cullin 1 RING-ligase) em Leishmania infantum

Abstract

The ubiquitin-proteasome system (UPS) is responsible for protein ubiquitination, which is essential for intracellular proteolysis in eukaryotes. E3 ligases are part of the UPS and play a key role in the ubiquitination process. They recognize and ubiquitinate substrates, targeting them for degradation by the proteasome or processing by deubiquitinase enzymes (DUBs). Cullin-RING-type E3 ligases (CRLs) are the largest and most studied class of mammalian E3s. CRLs are composed of SKP1, CUL1, RBX1, and an F-box protein that interacts with SKP1 through its F-box domain and recruits substrates for ubiquitination. In protozoan parasites that alternate between hosts, intracellular proteolysis is essential for parasitism. However, the SUP of many parasites remains unexplored, including trypanosomatids of the genus Leishmania. Individuals of this genus cause leishmaniasis, a set of diseases with different clinical manifestations (cutaneous, mucocutaneous and visceral forms) depending on the infecting species. Previous results from our group demonstrated that the LinfCRL1 complex is present in L. infantum regulating the cell cycle, and the interactions between its components were validated. In this study, the phylogenetic analysis of orthologous proteins from 12 Leishmania species returned two main clades, formed by the subgenera Viannia and Leishmania, corroborating the already known phylogenetic relationship, inferring the conservation of these genes in Leishmania. In addition, our study performed structural analyses and molecular dynamics simulation of the LinfCRL1 complex. The L. infantum proteins LinfSkp1, LinfCul1 and LinfRbx1 are structurally similar compared to their respective human orthologs: SKP1, CUL1 and RBX1. The interaction motifs of each protein are highly conserved. The similarity was quantified by the RMSD metric with results of 1.7, 4.8 and 7 Å, respectively for SKP1, CUL1 and RBX1. We assembled these proteins in a complex with the first 100 amino acids of the L. infantum F-box protein LinfFlp1. The structural prediction program AlphaFold2 returned a model with the same conformational pattern as the H. sapiens complex, suggesting the same cellular function. To visualize the behavior of the complex over time, we performed Molecular Dynamics (MD), revealing a mobility in LinfCul1, bringing LinfSkp1 closer to LinfRbx1. This movement has not yet been described in the H. sapiens ortholog. Collectively, these results are a significant step in the functional characterization of LymphCRLs in Leishmania, essential for understanding the biology of these parasites.