scholarly journals Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites

2021 ◽  
Vol 22 (11) ◽  
pp. 5709
Author(s):  
Mitzi Díaz-Hernández ◽  
Rosario Javier-Reyna ◽  
Izaid Sotto-Ortega ◽  
Guillermina García-Rivera ◽  
Sarita Montaño ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Entamoeba Histolytica ◽  
Posttranslational Modifications ◽  
Target Protein ◽  
Binding Motif ◽  
Docking Analysis ◽  
Membrane Activity ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus

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.

scholarly journals Protein SUMOylation is crucial for phagocytosis in Entamoeba histolytica trophozoites

2021 ◽  
Author(s):  
Mitzi Díaz-Hernández ◽  
Rosario Javier Reyna ◽  
Izaid Sotto-Ortega ◽  
Guillermina García-Rivera ◽  
Maricela Sarita Montaño ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Covalent Binding ◽  
3D Structure ◽  
Fine Tuning ◽  
Binding Motif ◽  
C Terminus ◽  
Protein Functions ◽  
Target Molecules

AbstractDuring 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 α−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 AbstractPhagocytosis is one of the main functions that Entamoeba histolyitica 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.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

scholarly journals Copper Induces Cytoplasmic Retention of Fission Yeast Transcription Factor Cuf1

2006 ◽  
Vol 5 (2) ◽  
pp. 277-292 ◽  
Author(s):  
Jude Beaudoin ◽  
Simon Labbé
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Nuclear Localization ◽  
Copper Transport ◽  
Nuclear Localization Sequence ◽  
Binding Motif ◽  
Physical Interaction ◽  
Dependent Manner ◽  
C Terminus ◽  
N Terminus

ABSTRACT Copper homeostasis within the cell is established and preserved by different mechanisms. Changes in gene expression constitute a way of maintaining this homeostasis. In Schizosaccharomyces pombe, the Cuf1 transcription factor is critical for the activation of copper transport gene expression under conditions of copper starvation. However, in the presence of elevated intracellular levels of copper, the mechanism of Cuf1 inactivation to turn off gene expression remains unclear. In this study, we provide evidence that inactivation of copper transport gene expression by Cuf1 is achieved through a copper-dependent, cytosolic retention of Cuf1. We identify a minimal nuclear localization sequence (NLS) between amino acids 11 to 53 within the Cuf1 N terminus. Deletion of this region and specific mutation of the Lys13, Arg16, Arg19, Lys24, Arg28, Lys45, Arg47, Arg50, and Arg53 residues to alanine within this putative NLS is sufficient to abrogate nuclear targeting of Cuf1. Under conditions of copper starvation, Cuf1 resides in the nucleus. However, in the presence of excess copper as well as silver ions, Cuf1 is sequestered in the cytoplasm, a process which requires the putative copper binding motif, 328Cys-X-Cys-X3-Cys-X-Cys-X2-Cys-X2-His342 (designated C-rich), within the C-terminal region of Cuf1. Deletion of this region and mutation of the Cys residues within the C-rich motif result in constitutive nuclear localization of Cuf1. By coexpressing the Cuf1 N terminus with its C terminus in trans and by using a two-hybrid assay, we show that these domains physically interact with each other in a copper-dependent manner. We propose a model wherein copper induces conformational changes in Cuf1 that promote a physical interaction between the Cuf1 N terminus and the C-rich motif in the C terminus that masks the NLS. Cuf1 is thereby sequestered in the cytosol under conditions of copper excess, thereby extinguishing copper transport gene expression.

The MDS-Associated Protein EVI1 Impairs Erythroid and Megakaryocytic Differentiation by Direct Interaction with GATA-1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3007-3007
Author(s):  
Leopoldo Laricchia-Robbio ◽  
Donglan Li ◽  
Raffaella Fazzina ◽  
Soumen Chakraborty ◽  
Maher Abdul Hay ◽  
...  
Keyword(s):  
Dna Binding ◽  
Reporter Gene ◽  
Chromosomal Abnormalities ◽  
Point Mutations ◽  
Binding Motif ◽  
Murine Bone Marrow ◽  
C Terminus ◽  
N Terminus ◽  
Reporter Gene Assays

Abstract EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in acute myeloid leukemia and myelodysplastic syndrome. This protein has two Zn finger domains containing 7 motifs at the N-terminus and 3 motifs at the C-terminus. The expression of this gene is a very poor prognostic marker and is associated with diseases characterized by erythroid and megakaryocytic defects. We have recently shown that the forced expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including fatal dyserythropoiesis, dysmegakaryopoiesis, and anemia. These lineages are regulated by the transcription factor GATA-1, a DNA-binding protein that in addition to erythrocytes and megakaryocytes exerts a strict control also on the differentiation of mast cells and eosinophils, on the basis of its expression and association with specific partners. In the present study, we used biochemical assays and in vitro culture to show that GATA-1 and the N-terminus of EVI1 are involved in the formation of a protein complex that is unable to regulate efficiently GATA-1-dependent promoters in reporter gene assays. EMSA studies with a GATA-1-specific probe indicate that EVI1 does not recognize and bind to the DNA probe but disrupts the DNA-binding of GATA-1. By deletion analysis and point mutations, we mapped the interaction between the proteins to two motifs in the proximal Zn finger domain of EVI1 and to the C-terminus Zn finger of GATA-1. Cys to Ala mutations in the two EVI1 motifs abrogate the interaction and restore the response of a promoter in reporter gene assays. We propose that the association between EVI1 and the DNA-binding motif of GATA-1 impairs efficient promoter binding by GATA-1 and the regulation of erythroid and megakaryocytic lineage. There studies suggest that the interaction surface between the two proteins could be an attractive target for the development of competing small molecules as a treatment in EVI1-associated leukemia.

scholarly journals Maturation of active zone assembly by Drosophila Bruchpilot

2009 ◽  
Vol 186 (1) ◽  
pp. 129-145 ◽  
Author(s):  
Wernher Fouquet ◽  
David Owald ◽  
Carolin Wichmann ◽  
Sara Mertel ◽  
Harald Depner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Drosophila Melanogaster ◽  
High Resolution ◽  
Active Zone ◽  
Synaptic Vesicles ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus ◽  
Dense Projections ◽  
Ca2 Channels

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

scholarly journals The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity

2016 ◽  
Vol 473 (2) ◽  
pp. 135-144 ◽  
Author(s):  
Glenn R. Masson ◽  
Olga Perisic ◽  
John E. Burke ◽  
Roger L. Williams
Keyword(s):  
Substrate Specificity ◽  
Tumour Suppressor ◽  
Membrane Activity ◽  
Intrinsically Disordered ◽  
C Terminus ◽  
N Terminus ◽  
Unstructured Regions

The unstructured regions found at the C-terminus of the tumour suppressor PTEN and the N-terminus PTEN-L can switch the enzymes' substrate specificity from soluble to membrane-embedded, and can also dramatically alter the enzymes' affinity for membranes.

GSDM family genes meet autophagy

2015 ◽  
Vol 469 (2) ◽  
pp. e5-e7 ◽  
Author(s):  
Masaru Tamura ◽  
Toshihiko Shiroishi
Keyword(s):  
Protein Function ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus ◽  
Autophagic Activity

Shi et al. provided a new insight of Gsdm family protein function. They reported that N- and C-terminus domain of Gsdm family protein interact each other. Moreover, the N-terminus domain of Gsdm family proteins has pro-autophagic activity, which is suppressed by the C-terminus domain.

scholarly journals Minor sequence modifications in temporin B cause drastic changes in antibacterial potency and selectivity by fundamentally altering membrane activity

2018 ◽  
Author(s):  
Giorgia Manzo ◽  
Philip M. Ferguson ◽  
V. Benjamin Gustilo ◽  
Tam T. Bui ◽  
Alex F. Drake ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Activity ◽  
Gram Positive Bacteria ◽  
C Terminus ◽  
N Terminus ◽  
Mechanism Of Interaction ◽  
Minor Sequence ◽  
Dynamics Simulations ◽  
Effective Translation

ABSTRACTAntimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required to understand their native function and for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMP activity. Relatively modest modifications to AMP primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Staphylococcus aureus while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.

Acentrosomal spindle morphogenesis is dependent on the unconventional TPX2 family protein TPXL3 and a Aurora kinase in Arabidopsis thaliana

2021 ◽  
Author(s):  
Xingguang Deng ◽  
Takumi Higaki ◽  
Honghui Lin ◽  
Yuh-Ru Julie Lee ◽  
Bo Liu
Keyword(s):  
Arabidopsis Thaliana ◽  
Nuclear Envelope ◽  
Aurora Kinase ◽  
Binding Motif ◽  
Mitotic Kinase ◽  
C Terminus ◽  
Family Protein ◽  
Dynamic Cell ◽  
Spindle Microtubules ◽  
Cycle Dependent

Abstract The α Aurora kinase serves as an essential mitotic regulator of spindle assembly in Arabidopsis thaliana. This indispensable function was thought to be dependent on an evolutionarily conserved targeting factor known as TPX2. However, our previous work indicated that the canonical TPX2 protein was dispensable but the plant specific TPX2 family protein TPXL3 became essential in A. thaliana. Here, we found that the TPXL3 polypeptide contains a microtubule-binding domain following the N-terminal Aurora-binding and activation motif, and its TPX2 signature domain with an importin-binding motif is followed by a novel short C-terminus. To test the hypothesis that TPXL3 dictated the essential mitotic function of α Aurora, we generated artificial microRNA lines in which TPXL3 expression was compromised. The amiR-TPXL3 mutants exhibited phenotypes of TPXL3 expression-correlated growth retardation. Due to the compromised TPXL3 expression, α Aurora delocalized from spindle microtubules and diffused in the cytoplasm. Such a phenotype was concomitant with defects in spindle morphogenesis as represented by disorganized spindle poles. A functional TPXL3-GFP fusion protein colocalized with α Aurora by exhibiting a highly dynamic cell cycle-dependent localization pattern highlighted by prominent appearance on the prophase nuclear envelope, spindle microtubules with biases towards poles, and reforming nuclear envelope during telophase and cytokinesis. However, TPXL3 was largely absent from phragmoplast microtubules that exhibited prominent γ-tubulin association. Finally, we found that the C-terminal domain was dispensable while other domains are required for its mitotic function. Hence, our discovery of the TPXL3 function shifted the TPX2-centered paradigm of mitotic kinase regulation in plants.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

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