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PLoS Pathogens Dec 2023Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have...
Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry.
Topics: Humans; Hemorrhagic Fever, Ebola; Ebolavirus; Calcium; Viral Envelope Proteins; Endosomes; Protein Conformation; Virus Internalization; Membrane Fusion; Hydrogen-Ion Concentration
PubMed: 38055723
DOI: 10.1371/journal.ppat.1011848 -
Frontiers in Cellular and Infection... 2022, the causative agent of human amoebiasis, exhibits a continuous membrane remodelling to exert its virulence properties. During this dynamic process, the Endosomal... (Review)
Review
, the causative agent of human amoebiasis, exhibits a continuous membrane remodelling to exert its virulence properties. During this dynamic process, the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is a key player, particularly in phagocytosis, a virulence hallmark of this parasite. In addition to ESCRT, other molecules contribute to membrane remodelling, including the EhADH adhesin, EhRabs, actin, and the lysobisphosphatidic acid (LBPA). The endocytosis of a prey or molecules induces membrane invaginations, resulting in endosome and multivesicular bodies (MVBs) formation for cargo delivery into lysosomes. Alternatively, some proteins are recycled or secreted. Most of these pathways have been broadly characterized in other biological systems, but poorly described in protozoan parasites. Here, we encompass 10 years of ESCRT research in , highlighting the role of the ESCRT-I and ESCRT-III components and the EhADH and EhVps4-ATPase accessory proteins during phagocytosis. In particular, EhADH exhibits a multifunctional role along the endocytic pathway, from cargo recognition to endosome maturation and lysosomal degradation. Interestingly, the interaction of EhADH with EhVps32 seems to shape a concurrent route to the conventional one for MVBs biogenesis, that could optimize their formation. Furthermore, this adhesin is secreted, but its role in this event remains under study. Other components from the endosomal pathway, such as EhVps23 and LBPA, are also secreted. A proteomic approach performed here, using an anti-LBPA antibody, revealed that some proteins related to membrane trafficking, cellular transport, cytoskeleton dynamics, and transcriptional and translational functions are secreted and associated to LBPA. Altogether, the accumulated knowledge around the ESCRT machinery in , points it out as a dynamic platform facilitating the interaction of molecules participating in different cellular events. Seen as an integrated system, ESCRTs lead to a better understanding of phagocytosis.
Topics: Humans; Entamoeba histolytica; Proteomics; Endosomes; Endosomal Sorting Complexes Required for Transport; Phagocytosis
PubMed: 36389174
DOI: 10.3389/fcimb.2022.855797 -
International Journal of Molecular... Jan 2017A mechanistic link between neuron-to-neuron transmission of secreted amyloid and propagation of protein malconformation cytopathology and disease has recently been... (Review)
Review
A mechanistic link between neuron-to-neuron transmission of secreted amyloid and propagation of protein malconformation cytopathology and disease has recently been uncovered in animal models. An enormous interest in the unconventional secretion of amyloids from neurons has followed. Amphisomes and late endosomes are the penultimate maturation products of the autophagosomal and endosomal pathways, respectively, and normally fuse with lysosomes for degradation. However, under conditions of perturbed membrane trafficking and/or lysosomal deficiency, prelysosomal compartments may instead fuse with the plasma membrane to release any contained amyloid. After a brief introduction to the endosomal and autophagosomal pathways, we discuss the evidence for autophagosomal secretion (exophagy) of amyloids, with a comparative emphasis on Aβ and α-synuclein, as luminal and cytosolic amyloids, respectively. The ESCRT-mediated import of cytosolic amyloid into late endosomal exosomes, a known vehicle of transmission of macromolecules between cells, is also reviewed. Finally, mechanisms of lysosomal dysfunction, deficiency, and exocytosis are exemplified in the context of genetically identified risk factors, mainly for Parkinson's disease. Exocytosis of prelysosomal or lysosomal organelles is a last resort for clearance of cytotoxic material and alleviates cytopathy. However, they also represent a vehicle for the concentration, posttranslational modification, and secretion of amyloid seeds.
Topics: Amyloid; Animals; Autophagy; Cell Compartmentation; Endosomes; Humans; Lysosomes; alpha-Synuclein
PubMed: 28124989
DOI: 10.3390/ijms18010227 -
Human Genetics Mar 2023CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to...
CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR-Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development.
Topics: Animals; Humans; Actins; DNA-Binding Proteins; Endosomes; HEK293 Cells; Lectins, C-Type; Membrane Proteins; Monosaccharide Transport Proteins; Nuclear Proteins; Protein Transport; Transcription Factors; Ubiquitin-Protein Ligases; Ubiquitins; Zebrafish
PubMed: 36538041
DOI: 10.1007/s00439-022-02511-3 -
Methods in Cell Biology 2021Endosomal microautophagy (eMI) is a type of autophagy that allows for the selective uptake and degradation of cytosolic proteins in late endosome/multi-vesicular bodies...
Endosomal microautophagy (eMI) is a type of autophagy that allows for the selective uptake and degradation of cytosolic proteins in late endosome/multi-vesicular bodies (LE/MVB). This process starts with the recognition of a pentapeptide amino acid KFERQ-like targeting motif in the substrate protein by the hsc70 chaperone, which then enables binding and subsequent uptake of the protein into the LE/MVB compartment. The recognition of a KFERQ-like motif by hsc70 is the same initial step in chaperone-mediated autophagy (CMA), a form of selective autophagy that degrades the hsc70-targeted proteins in lysosomes in a LAMP-2A dependent manner. The shared step of substrate recognition by hsc70, originally identified for CMA, makes it now necessary to differentiate between the two pathways. Here, we detail biochemical and imaging-based methods to track eMI activity in vitro with isolated LE/MVBs and in cells in culture using fluorescent reporters and highlight approaches to distinguish whether a protein is a substrate of eMI or CMA.
Topics: Animals; Autophagy; Endosomes; HSC70 Heat-Shock Proteins; Lysosomes; Microautophagy
PubMed: 34225914
DOI: 10.1016/bs.mcb.2020.10.009 -
Autophagy 2018Rab2 is a conserved Rab GTPase with a well-established role in secretory pathway function and phagocytosis. Here we demonstrate that Drosophila Rab2 is recruited to late...
Rab2 is a conserved Rab GTPase with a well-established role in secretory pathway function and phagocytosis. Here we demonstrate that Drosophila Rab2 is recruited to late endosomal membranes, where it controls the fusion of LAMP-containing biosynthetic carriers and lysosomes to late endosomes. In contrast, the lysosomal GTPase Gie/Arl8 is only required for late endosome-lysosome fusion, but not for the delivery of LAMP to the endocytic pathway. We also find that Rab2 is required for the fusion of autophagosomes to the endolysosomal pathway, but not for the biogenesis of lysosome-related organelles. Surprisingly, Rab2 does not rely on HOPS-mediated vesicular fusion for recruitment to late endosomal membranes. Our work suggests that Drosophila Rab2 is a central regulator of the endolysosomal and macroautophagic/autophagic pathways by controlling the major heterotypic fusion processes at the late endosome.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Endocytosis; Endosomes; Gene Expression Regulation; Green Fluorescent Proteins; Intracellular Membranes; Lysosomes; Membrane Fusion; Membrane Proteins; Multiprotein Complexes; Mutation; Phenotype; Proteolysis; Vacuoles
PubMed: 29940804
DOI: 10.1080/15548627.2018.1458170 -
PLoS Pathogens Aug 2023Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms...
Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.
Topics: Humans; Sandfly fever Naples virus; Endocytosis; Endosomes; Vacuoles; Virus Internalization; Hydrogen-Ion Concentration
PubMed: 37578957
DOI: 10.1371/journal.ppat.1011562 -
EMBO Reports Aug 2023The ER regulates the spatiotemporal organization of endolysosomal systems by membrane contact. In addition to tethering via heterotypic interactions on both organelles,...
The ER regulates the spatiotemporal organization of endolysosomal systems by membrane contact. In addition to tethering via heterotypic interactions on both organelles, we present a novel ER-endosome tethering mechanism mediated by homotypic interactions. The single-pass transmembrane protein SCOTIN is detected in the membrane of the ER and endosomes. In SCOTIN-knockout (KO) cells, the ER-late endosome contacts are reduced, and the perinuclear positioning of endosomes is disturbed. The cytosolic proline-rich domain (PRD) of SCOTIN forms homotypic assemblies in vitro and is necessary for ER-endosome membrane tethering in cells. A region of 28 amino acids spanning 150-177 within the SCOTIN PRD is essential to elicit membrane tethering and endosomal dynamics, as verified by reconstitution in SCOTIN-KO cells. The assembly of SCOTIN (PRD) is sufficient to mediate membrane tethering, as purified SCOTIN (PRD), but not SCOTIN (PRDΔ150-177), brings two different liposomes closer in vitro. Using organelle-specific targeting of a chimeric PRD domain shows that only the presence on both organellar membranes enables the ER-endosome membrane contact, indicating that the assembly of SCOTIN on heterologous membranes mediates organelle tethering.
Topics: Intracellular Membranes; Endoplasmic Reticulum; Membrane Proteins; Endosomes
PubMed: 37377038
DOI: 10.15252/embr.202256538 -
Proceedings of the National Academy of... Sep 2023The canonical view of G protein-coupled receptor (GPCR) function is that receptor trafficking is tightly coupled to signaling. GPCRs remain on the plasma membrane (PM)...
The canonical view of G protein-coupled receptor (GPCR) function is that receptor trafficking is tightly coupled to signaling. GPCRs remain on the plasma membrane (PM) at the cell surface until they are activated, after which they are desensitized and internalized into endosomal compartments. This canonical view presents an interesting context for proton-sensing GPCRs because they are more likely to be activated in acidic endosomal compartments than at the PM. Here, we show that the trafficking of the prototypical proton-sensor GPR65 is fully uncoupled from signaling, unlike that of other known mammalian GPCRs. GPR65 internalizes and localizes to early and late endosomes, from where they signal at steady state, irrespective of extracellular pH. Acidic extracellular environments stimulate receptor signaling at the PM in a dose-dependent manner, although endosomal GPR65 is still required for a full signaling response. Receptor mutants that were incapable of activating cAMP trafficked normally, internalize and localize to endosomal compartments. Our results show that GPR65 is constitutively active in endosomes, and suggest a model where changes in extracellular pH reprograms the spatial pattern of receptor signaling and biases the location of signaling to the cell surface.
Topics: Animals; Protons; Cell Membrane; Endosomes; Signal Transduction; Mammals
PubMed: 37722051
DOI: 10.1073/pnas.2302823120 -
Frontiers in Cellular and Infection... 2023The African swine fever virus (ASFV) is strongly dependent on an intact endocytic pathway and a certain cellular membrane remodeling for infection, possibly regulated by...
The African swine fever virus (ASFV) is strongly dependent on an intact endocytic pathway and a certain cellular membrane remodeling for infection, possibly regulated by the endosomal sorting complexes required for transport (ESCRT). The ESCRT machinery is mainly involved in the coordination of membrane dynamics; hence, several viruses exploit this complex and its accessory proteins VPS4 and ALIX for their own benefit. In this work, we found that shRNA-mediated knockdown of VPS4A decreased ASFV replication and viral titers, and this silencing resulted in an enhanced expression of ESCRT-0 component HRS. ASFV infection slightly increased HRS expression but not under VPS4A depletion conditions. Interestingly, VPS4A silencing did not have an impact on ALIX expression, which was significantly overexpressed upon ASFV infection. Further analysis revealed that ALIX silencing impaired ASFV infection at late stages of the viral cycle, including replication and viral production. In addition to ESCRT, the accessory protein ALIX is involved in endosomal membrane dynamics in a lysobisphosphatydic acid (LBPA) and Ca-dependent manner, which is relevant for intraluminal vesicle (ILV) biogenesis and endosomal homeostasis. Moreover, LBPA interacts with NPC2 and/or ALIX to regulate cellular cholesterol traffic, and would affect ASFV infection. Thus, we show that LBPA blocking impacted ASFV infection at both early and late infection, suggesting a function for this unconventional phospholipid in the ASFV viral cycle. Here, we found for the first time that silencing of VPS4A and ALIX affects the infection later on, and blocking LBPA function reduces ASFV infectivity at early and later stages of the viral cycle, while ALIX was overexpressed upon infection. These data suggested the relevance of ESCRT-related proteins in ASFV infection.
Topics: Swine; Animals; Endosomal Sorting Complexes Required for Transport; African Swine Fever Virus; Calcium-Binding Proteins; Endosomes; Endocytosis
PubMed: 38125905
DOI: 10.3389/fcimb.2023.1163569