A noncanonical GATA transcription factor of <i>Entamoeba histolytica</i> modulates genes involved in phagocytosis

Huerta, Miriam; Reyes, Luz M.; Garcı́a-Rivera, Guillermina; Bañuelos, Cecilia; Betanzos, Abigail; Díaz-Hernández, Mitzi; Galindo, Ausencio; Bolaños, Jeni; Cárdenas, Helios; Azuara-Licéaga, Elisa; Chávez‐Munguía, Bibiana; Orozco, Esther

doi:10.1111/mmi.14592

Cited by 4 publications

(3 citation statements)

References 83 publications

(112 reference statements)

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“…The full-length gene was cloned in pCold II DNA plasmid, which conferred a histidine tag [ 68 ]. As a positive control of the reaction, we use primers to amplify the Ehgata gene [ 69 ]. Two negative controls were included: in the first, water was used instead of template; and in the second, the DNAse was omitted in the mixture.…”

Section: Methodsmentioning

confidence: 99%

Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites

Díaz-Hernández

Javier-Reyna

Sotto-Ortega

et al. 2021

IJMS

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Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.

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Section: Methodsmentioning

confidence: 99%

Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites

Díaz-Hernández

Javier-Reyna

Sotto-Ortega

et al. 2021

IJMS

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“…LARP1, EIF2, GATA4 and RICTOR have been shown to play roles in cell phagocytosis and degradation, indicating their possible role in PCSK9-mediated efferocytosis. [41][42][43][44][45] Collectively, our proteomics analyses in Disease and Bio Functions suggest that Pep2-8 treatment significantly inhibits ROS synthesis and generation, cell death of muscle cells, cell immune response and necrosis (unprogrammed death of cells and living tissue) compared with MOB 1-8 (Figure 6F). This analysis also implies that Pep2-8 may inhibit cell death and apoptosis in tumor/cancer cells, indicating that PCSK9 inhibition may provide benefit to vascular aging but potentially predisposing to neoplasms, a possibility that will require careful evaluation.…”

Section: Database Analysis Of Pcsk9 In Disease and Biological Functionsmentioning

confidence: 82%

PCSK9 attenuates efferocytosis in endothelial cells and promotes vascular aging

Liu¹,

Wu²,

Stolarz³

et al. 2023

Theranostics

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Aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that binds to low-density lipoprotein receptors. Efferocytosis is the process by which phagocytes remove apoptotic cells. Both PCSK9 and efferocytosis play important roles in regulating redox biology and inflammation, the key factors contributing to vascular aging. This study was designed to investigate the impact of PCSK9 on efferocytosis in endothelial cells (ECs) and its implications in vascular aging. Methods and Results: Studies were performed in primary human aortic ECs (HAECs) and primary mouse aortic ECs (MAECs) isolated from male wild-type (WT) and PCSK9 -/- mice, and in young and aged mice treated with saline or the PCSK9 inhibitor Pep2-8. Our findings include that recombinant PCSK9 protein induces defective efferocytosis and aging marker senescence-associated-β-galactosidase (SA-β-gal) expression in ECs, while PCSK9 -/- restores efferocytosis and inhibits SA-β-gal activity. Further studies in aged mice showed that endothelial deficiency of MerTK, a critical receptor for efferocytosis that allows phagocytes to detect the presence of apoptotic cells, may be an indicator of vascular dysfunction in the aortic arch. Pep2-8 treatment markedly restored efferocytosis in endothelium from the aged mice. A proteomics study in the aortic arch from aged mice revealed that Pep2-8 administration significantly downregulates expression of NOX4, MAPK subunits, NF-κB, and secretion of pro-inflammatory cytokines, all known to promote vascular aging. Immunofluorescent staining showed that Pep2-8 administration upregulates expression of eNOS and downregulates expression of pro-IL-1β, NF-κB and p22 phox compared to saline treated group. Conclusions: These findings provide initial evidence for the ability of aortic ECs to accomplish efferocytosis and argue for a role of PCSK9 in attenuating EC efferocytosis, thereby leading to vascular dysfunction and acceleration in vascular aging.

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“…The full-length gene was cloned in pCold II DNA plasmid, which conferred a histidine tag (67). As positive control we use primers to amplify the EhGATA gene (68).…”

Section: Cloning Of the E Histolytica Sumo Gene (Ehsumo)mentioning

confidence: 99%

Protein SUMOylation is crucial for phagocytosis inEntamoeba histolyticatrophozoites

Díaz-Hernández

Javier-Reyna

Ortega-Soto

et al. 2021

Preprint

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During phagocytosis, a key event in the virulence of the protozoan Entamoeba histolytica , several molecules in concert contact the target, generate pseudopodia, and internalize and digest the ingested prey. Posttranslational modifications provide proteins the timing and signaling to intervene in these processes. SUMOylation is a posttranslational modification that in several systems grants a fine tuning for protein functions, protein interactions and cellular location, but it has not been studied in E. histolytica. In this paper, we characterized the E. histolytica SUMO gene and its product (EhSUMO) and elucidated the EhSUMO 3D-structure. Furthermore, here we studied the relevance of SUMOylation in phagocytosis, particularly in its association with EhADH (an ALIX family protein) and EhVps32 (a protein of the ESCRT-III complex), both involved in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates other SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machineries. Confocal microscopy assays, using a-EhSUMO antibodies disclosed a remarkable membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH, and immunoprecipitation and immunofluorescence assays revealed that the EhADH-EhSUMO association increased during phagocytosis, whereas the EhVps32-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. Author’s Abstract Phagocytosis is one of the main functions that Entamoeba histolytica trophozoites carry out during the invasion to the host. Many proteins are involved in this fascinating event, in which the plasmatic membrane undergoes to multiple and speedy changes. Posttraductional modifications activate proteins in the precise time that they must get involved. SUMOylation, that consists in the non-covalent binding of SUMO protein with target molecules, is one of the main changes suffered by proteins in order to enable them to participate in cellular functions. SUMOylation had not been studied in E. histolytica nor in phagocytosis, and our working hypothesis is that this event is deeply engaged in the ingestion of target molecules and cells. The results of this paper prove the presence of an intronless bona fide EhSUMO gene encoding for a predicted 12.6 kDa protein that is actively involved in phagocytosis. Silencing of the EhSUMO gene affected the rate of phagocytosis and interfered with the EhADH and EhVps32 function, two proteins involved in phagocytosis, strongly supporting the importance of SUMOylation in this event.

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A noncanonical GATA transcription factor of Entamoeba histolytica modulates genes involved in phagocytosis

Cited by 4 publications

References 83 publications

Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites

Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites

PCSK9 attenuates efferocytosis in endothelial cells and promotes vascular aging

Protein SUMOylation is crucial for phagocytosis inEntamoeba histolyticatrophozoites

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