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In humans and Drosophila melanogaster, the functional convergence of the endosomal sorting complex required for transport (ESCRT) machinery that is in charge of selecting ubiquitinated proteins for sorting into multivesicular bodies, and the retromer, that is the complex responsible for protein recycling to the plasma membrane and Golgi apparatus. ESCRT and retromer complexes are codependent for protein sorting recycling, degradation, and secretion. In this article, we studied the EhVps35 C isoform (referred to as EhVps35), that is the central member of the Entamoeba histolytica retromer, and its relation with the ESCRT machinery during sorting and protein recycling events and their involvement virulence. Our findings revealed that EhVps35 interacts with at least 300 proteins that participate in multiple cellular processes. Laser confocal and transmission electronic microscopy images, as well as secretion assays, revealed that EhVps35 is secreted in vesicles together with EhVps23 and EhADH (both ESCRT machinery proteins). In addition, immunoprecipitation, immunofluorescence, and molecular docking assays revealed the relationship among EhVps35 and other ESCRT machinery proteins. Red blood cell stimulus increased EhVps35 secretion, and the knockdown of the Ehvps35 gene in trophozoites reduced their capacity to migrate and invade tissues. This also impacts the cellular localization of ubiquitin, EhVps23 (ESCRT-I), and EhVps32 (ESCRT-III) proteins, strongly suggesting their functional relationship. Our results, taken together, give evidence that EhVps35 is a key factor in E. histolytica virulence mechanisms and that it, together with the ESCRT machinery components and other regulatory proteins, is involved in vesicle trafficking, secretion, migration, and cell proliferation.

The retromer is a highly conserved eukaryotic complex formed by the cargo selective complex (CSC) and the sorting nexin (SNX) dimer subcomplexes. Its function is protein recycling and recovery from the endosomes to conduct the target molecules to the trans-Golgi network or the plasma membrane. The protozoan responsible for human amoebiasis, Entamoeba histolytica, exhibits an active membrane movement and voracious phagocytosis, events in which the retromer may be fully involved. In this work, we studied the structure of EhVps35 the central member of the CSC retromeric subcomplex as it binds EhVps26 and EhVps29, the other two CSC members, allowing the position of the retromer in the membranes. We also studied the EhVps35 role in the recycling of virulence proteins, particularly those involved in phagocytosis. Confocal microscopy assays revealed that EhVps35 is located in the plasmatic and endosomal membranes and in the phagocytic cups and channels. In addition, it follows the target cell from the moment it is in contact with the trophozoites. Molecular docking analyses, immunoprecipitation assays, and microscopy studies revealed that EhVps35 interacts with the EhADH, Gal/GalNac lectin, and actin proteins. In addition, experimental evidence indicated that it recycles surface proteins, particularly EhADH and Gal/GalNac proteins, two molecules highly involved in virulence. Knockdown of the Ehvps35 gene induced a decrease in protein recycling, as well as impairments in the efficiency of adhesion and the rate of phagocytosis. The actin cytoskeleton was deeply affected by the Ehvps35 gene knockdown. In summary, our results revealed the participation of EhVps35 in protein recycling and phagocytosis. Furthermore, altogether, our results demonstrated the concert of finely regulated molecules, including EhVps35, EhADH, Gal/GalNac lectin, and actin, in the phagocytosis of E. histolytica.

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1–400 aa), Linker (246–446 aa) and Adh (444–687 aa) to evaluate their role in virulence. The TrophozBro11–400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226–446 also increased the virulence properties, but with lower effect than the TrophozBro11–400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444–687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes.

Abstract Oncogenic protein E6 from Human Papilloma Virus 16 (HPV-16) mediates the degradation of Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1), throughout the interaction of its protein binding motif (PBM) with the Discs-large homologous regions 1 (PDZ1) domain of MAG1-1. Generic variation in the E6 gene that translates to changes in the protein’s amino acidic sequence modifies the interaction of E6 with the cellular protein MAGI-1. MAGI-1 is a scaffolding protein found at tight junctions of epithelial cells, where it interacts with a variety of proteins regulating signaling pathways. MAGI-1 is a multidomain protein containing two WW (rsp-domain-9), one guanylate kinase-like, and six PDZ domains. PDZ domains played an important role in the function of MAGI-1 and served as targets for several viral proteins including the HPV-16 E6. The aim of this work was to evaluate, with an in silico approach, employing molecular dynamics simulation and protein–protein docking, the interaction of the intragenic variants E-G350 (L83V), E-C188/G350 (E29Q/L83V), E-A176/G350 (D25N/L83V), E6-AAa (Q14H/H78Y/83V) y E6-AAc (Q14H/I27RH78Y/L83V) and E6-reference of HPV-16 with MAGI-1. We found that variants E-G350, E-C188/G350, E-A176/G350, AAa and AAc increase their affinity to our two models of MAGI-1 compared to E6-reference.

Lymph node metastasis (LNM) is an important prognostic factor in cervical cancer (CC). In early stages, the risk of LNM is approximately 3.7 to 21.7%, and the 5-year overall survival decreases from 80% to 53% when metastatic disease is identified in the lymph nodes. Few reports have analyzed the relationship between miRNA expression and the presence of LNM. The aim of this study was to identify a subset of miRNAs related to LNM in early-stage CC patients. Formalin-fixed paraffin-embedded tissue blocks were collected from patients with early-stage CC treated by radical hysterectomy with lymphadenectomy. We analyzed samples from two groups of patients—one group with LNM and the other without LNM. Global miRNA expression was identified by microarray analysis, and cluster analysis was used to determine a subset of miRNAs associated with LNM. Microarray expression profiling identified a subset of 36 differentially expressed miRNAs in the two groups (fold change (FC) ≥ 1.5 and p < 0.01). We validated the expression of seven miRNAs; miR-487b, miR-29b-2-5p, and miR-195 were underexpressed, and miR-92b-5p, miR-483-5p, miR-4534, and miR-548ac were overexpressed according to the microarray experiments. This signature exhibited prognostic value for identifying early-stage CC patients with LNM. These findings may help detect LNM that cannot be observed in imaging studies.

By their active movement and voraux phagocytosis, the trophozoites of Entamoeba histolytica constitute an excellent system to investigate the dynamics of the Endosomal Sorting Complex Required for Transport (ESCRT) protein interactions through phagocytosis. Here, we studied the proteins forming the E. histolytica ESCRT-II complex and their relationship with other phagocytosis-involved molecules. Bioinformatics analysis predicted that EhVps22, EhVps25, and EhVps36 are E. histolytica bona fide orthologues of the ESCRT-II protein families. Recombinant proteins and specific antibodies revealed that ESCRT-II proteins interact with each other, with other ESCRT proteins, and phagocytosis-involved molecules, such as the adhesin (EhADH). Laser confocal microscopy, pull-down assays, and mass spectrometry analysis disclosed that during phagocytosis, ESCRT-II accompanies the red blood cells (RBCs) from their attachment to the trophozoites until their arrival to multivesicular bodies (MVBs), changing their interactive patterns according to the time and place of the process. Knocked-down trophozoites in the Ehvps25 gene presented a 50% lower rate of phagocytosis than the controls and lower efficiency to adhere RBCs. In conclusion, ESCRT-II interacts with other molecules during prey contact and conduction throughout the phagocytic channel and trophozoites membranous system. ESCRT-II proteins are members of the protein chain during vesicle trafficking and are fundamental for the continuity and efficiency of phagocytosis.

The endosomal sorting complex required for transport (ESCRT) is formed by ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III complexes, and accessory proteins. It conducts vesicular trafficking in eukaryotes through the formation of vesicles and membrane fission and fusion events. The trophozoites of Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents an active membrane movement in basal state that increases during phagocytosis and tissue invasion. ESCRT-III complex has a pivotal role during these events, but ESCRT-0, ESCRT-I and ESCRT-II have been poorly studied. Here, we unveiled the E. histolytica ESCRT-I complex and its implication in vesicular trafficking and phagocytosis, as well as the molecular relationships with other phagocytosis-involved molecules. We found a gene encoding for a putative EhVps23 protein with the ubiquitin-binding and Vps23 core domains. In basal state, it was in the plasma membrane, cytoplasmic vesicles and multivesicular bodies, whereas during phagocytosis it was extensively ubiquitinated and detected in phagosomes and connected vesicles. Docking analysis, immunoprecipitation assays and microscopy studies evidenced its interaction with EhUbiquitin, EhADH, EhVps32 proteins, and the lysobisphosphatidic acid phospholipid. The knocking down of the Ehvps23 gene resulted in lower rates of phagocytosis. Our results disclosed the concert of finely regulated molecules and vesicular structures participating in vesicular trafficking-related events with a pivotal role of EhVps23.