scholarly journals ESCRT-dependent membrane repair negatively regulates pyroptosis downstream of GSDMD activation

Science ◽  
2018 ◽  
Vol 362 (6417) ◽  
pp. 956-960 ◽  
Author(s):  
Sebastian Rühl ◽  
Kateryna Shkarina ◽  
Benjamin Demarco ◽  
Rosalie Heilig ◽  
José Carlos Santos ◽  
...  
Keyword(s):  
Calcium Influx ◽  
Inflammasome Activation ◽  
Membrane Repair ◽  
Cellular Survival ◽  
Endosomal Sorting ◽  
Membrane Pores ◽  
Escrt Machinery ◽  
Inflammatory Caspases ◽  
Survival Mechanisms ◽  
Insight Into

Pyroptosis is a lytic form of cell death that is induced by inflammatory caspases upon activation of the canonical or noncanonical inflammasome pathways. These caspases cleave gasdermin D (GSDMD) to generate an N-terminal GSDMD fragment, which executes pyroptosis by forming membrane pores. We found that calcium influx through GSDMD pores serves as a signal for cells to initiate membrane repair by recruiting the endosomal sorting complexes required for transport (ESCRT) machinery to damaged membrane areas, such as the plasma membrane. Inhibition of the ESCRT-III machinery strongly enhances pyroptosis and interleukin-1β release in both human and murine cells after canonical or noncanonical inflammasome activation. These results not only attribute an anti-inflammatory role to membrane repair by the ESCRT-III system but also provide insight into general cellular survival mechanisms during pyroptosis.

scholarly journals The VPS-20 subunit of the endosomal sorting complex ESCRT-III exhibits an open conformation in the absence of upstream activation

2015 ◽  
Vol 466 (3) ◽  
pp. 625-637 ◽  
Author(s):  
Amber L. Schuh ◽  
Michael Hanna ◽  
Kyle Quinney ◽  
Lei Wang ◽  
Ali Sarkeshik ◽  
...  
Keyword(s):  
Membrane Repair ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Cell Extracts ◽  
Open Conformation ◽  
Membrane Reorganization ◽  
Plasma Membrane Repair ◽  
Retroviral Budding ◽  
Vacuolar Protein

Members of the endosomal sorting complex required for transport (ESCRT) machinery function in membrane remodelling processes during multivesicular endosome (MVE) biogenesis, cytokinesis, retroviral budding and plasma membrane repair. During luminal vesicle formation at endosomes, the ESCRT-II complex and the ESCRT-III subunit vacuolar protein sorting (VPS)-20 play a specific role in regulating assembly of ESCRT-III filaments, which promote vesicle scission. Previous work suggests that Vps20 isoforms, like other ESCRT-III subunits, exhibits an auto-inhibited closed conformation in solution and its activation depends on an association with ESCRT-II specifically at membranes [1]. However, we show in the present study that Caenorhabditis elegans ESCRT-II and VPS-20 interact directly in solution, both in cytosolic cell extracts and in using recombinant proteins in vitro. Moreover, we demonstrate that purified VPS-20 exhibits an open extended conformation, irrespective of ESCRT-II binding, in contrast with the closed auto-inhibited architecture of another ESCRT-III subunit, VPS-24. Our data argue that individual ESCRT-III subunits adopt distinct conformations, which are tailored for their specific functions during ESCRT-mediated membrane reorganization events.

Growing functions of the ESCRT machinery in cell biology and viral replication

2017 ◽  
Vol 45 (3) ◽  
pp. 613-634 ◽  
Author(s):  
Edward J. Scourfield ◽  
Juan Martin-Serrano
Keyword(s):  
Cell Biology ◽  
Modular System ◽  
Membrane Repair ◽  
Endosomal Sorting ◽  
Cellular Processes ◽  
Functional Connections ◽  
Escrt Machinery ◽  
Viral Budding ◽  
Spatiotemporal Regulation ◽  
Plasma Membrane Repair

The vast expansion in recent years of the cellular processes promoted by the endosomal sorting complex required for transport (ESCRT) machinery has reinforced its identity as a modular system that uses multiple adaptors to recruit the core membrane remodelling activity at different intracellular sites and facilitate membrane scission. Functional connections to processes such as the aurora B-dependent abscission checkpoint also highlight the importance of the spatiotemporal regulation of the ESCRT machinery. Here, we summarise the role of ESCRTs in viral budding, and what we have learned about the ESCRT pathway from studying this process. These advances are discussed in the context of areas of cell biology that have been transformed by research in the ESCRT field, including cytokinetic abscission, nuclear envelope resealing and plasma membrane repair.

scholarly journals Plasma membrane integrity in health and disease: significance and therapeutic potential

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Catarina Dias ◽  
Jesper Nylandsted
Keyword(s):  
Plasma Membrane ◽  
Therapeutic Potential ◽  
Calcium Influx ◽  
Membrane Integrity ◽  
Cell Types ◽  
Physical Parameters ◽  
Membrane Repair ◽  
Plasma Membrane Integrity ◽  
Repair Mechanisms ◽  
And Function

AbstractMaintenance of plasma membrane integrity is essential for normal cell viability and function. Thus, robust membrane repair mechanisms have evolved to counteract the eminent threat of a torn plasma membrane. Different repair mechanisms and the bio-physical parameters required for efficient repair are now emerging from different research groups. However, less is known about when these mechanisms come into play. This review focuses on the existence of membrane disruptions and repair mechanisms in both physiological and pathological conditions, and across multiple cell types, albeit to different degrees. Fundamentally, irrespective of the source of membrane disruption, aberrant calcium influx is the common stimulus that activates the membrane repair response. Inadequate repair responses can tip the balance between physiology and pathology, highlighting the significance of plasma membrane integrity. For example, an over-activated repair response can promote cancer invasion, while the inability to efficiently repair membrane can drive neurodegeneration and muscular dystrophies. The interdisciplinary view explored here emphasises the widespread potential of targeting plasma membrane repair mechanisms for therapeutic purposes.

scholarly journals Listeriolysin O: from bazooka to Swiss army knife

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160222 ◽  
Author(s):  
Suzanne E. Osborne ◽  
John H. Brumell
Keyword(s):  
Systemic Disease ◽  
Membrane Damage ◽  
Host Cells ◽  
Listeriolysin O ◽  
Tissue Destruction ◽  
Membrane Repair ◽  
Membrane Pores ◽  
Bacterial Dissemination ◽  
Versatile Tool ◽  
Cell Spread

Listeria monocytogenes ( Lm ) is a Gram-positive facultative intracellular pathogen. Infections in humans can lead to listeriosis, a systemic disease with a high mortality rate. One important mechanism of Lm dissemination involves cell-to-cell spread after bacteria have entered the cytosol of host cells. Listeriolysin O (LLO; encoded by the hly gene) is a virulence factor present in Lm that plays a central role in the cell-to-cell spread process. LLO is a member of the cholesterol-dependent cytolysin (CDC) family of toxins that were initially thought to promote disease largely by inducing cell death and tissue destruction—essentially acting like a ‘bazooka’. This view was supported by structural studies showing CDCs can form large pores in membranes. However, it is now appreciated that LLO has many subtle activities during Lm infection of host cells, and many of these likely do not involve large pores, but rather small membrane perforations. It is also appreciated that membrane repair pathways of host cells play a major role in limiting membrane damage by LLO and other toxins. LLO is now thought to represent a ‘Swiss army knife’, a versatile tool that allows Lm to induce many membrane alterations and cellular responses that promote bacterial dissemination during infection. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

scholarly journals Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

2015 ◽  
Vol 89 (23) ◽  
pp. 11750-11760 ◽  
Author(s):  
Timothy K. Soh ◽  
Sean P. J. Whelan
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Proteins ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Viral Particles ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Late Domains

ABSTRACTVesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.IMPORTANCEAssembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a “late domain” that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

scholarly journals cAMP metabolism controls caspase-11 inflammasome activation and pyroptosis in sepsis

2019 ◽  
Vol 5 (5) ◽  
pp. eaav5562 ◽  
Author(s):  
Ruochan Chen ◽  
Ling Zeng ◽  
Shan Zhu ◽  
Jiao Liu ◽  
Herbert J. Zeh ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Inflammatory Responses ◽  
Poly I:C ◽  
Inflammasome Activation ◽  
Proof Of Concept ◽  
Potential Therapeutic Target ◽  
Camp Metabolism ◽  
Camp Hydrolysis ◽  
Insight Into

The ability of cytosolic lipopolysaccharide (LPS) to activate caspase-11–dependent nonclassical inflammasome is intricately controlled to avoid excessive inflammatory responses. However, very little is known about the regulatory role of various metabolic pathways in the control of caspase-11 activation. Here, we demonstrate that l-adrenaline can act on receptor ADRA2B to inhibit the activation of the caspase-11 inflammasome by cytosolic LPS or Escherichia coli infection in macrophages. l-adrenaline–induced cAMP production via the enzyme ADCY4 promotes protein kinase A (PKA) activation, which then blocks the caspase-11–mediated proteolytic maturation of interleukin-1β, gasdermin D (GSDMD) cleavage, and consequent DAMP release. Inhibition of PDE8A-mediated cAMP hydrolysis limits caspase-11 inflammasome activation and pyroptosis in macrophages. Consequently, pharmacological modulation of the ADRA2B-ADCY4-PDE8A-PKA axis, knockout of caspase-11 (Casp11−/−), or Gsdmd inactivation (GsdmdI105N/I105N) similarly protects against LPS-induced lethality in poly(I:C)-primed mice. Our results provide previously unidentified mechanistic insight into immune regulation by cAMP and represent a proof of concept that immunometabolism constitutes a potential therapeutic target in sepsis.

scholarly journals Nanobody-Mediated Inhibition of P2X7 on Microglia Improves Stroke Outcome

2021 ◽  
Author(s):  
Maximilian Wilmes ◽  
Carolina Pinto Espinoza ◽  
Peter Ludewig ◽  
Arthur Liesz ◽  
Annette Nicke ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Transgenic Mice ◽  
Tissue Damage ◽  
Calcium Influx ◽  
Atp Release ◽  
Artery Occlusion ◽  
Membrane Pores ◽  
Caspase 1 ◽  
Ischemic Tissue

Abstract BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple brain disease models. However, tools for the selective and efficient targeting of these receptors are scarce. The new development of P2X7-specific nanobodies (nbs) enables us to effectively block the P2X7-channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wildtype and P2X7-transgenic mice was used as a model for ischemic stroke. ATP release was assessed in transgenic ATP sensor mice. Stroke size was measured without treatment and after injection of P2X7-specific nbs i.v. and i.c.v. directly before tMCAO-surgery. P2X7-GFP expressing transgenic mice were used to show immunhistochemically P2X7 distribution in the brain. In vitro cultured microglia were used to investigate calcium-influx, pore-formation via DAPI uptake, caspase 1 activation and IL-1b release after incubation with P2X7-specific nbs. ResultsATP sensor mice showed an increase of ATP-release in the ischemic hemisphere compared to the contralateral hemisphere or sham mice up to 24 h after stroke. We could further verify the role of the ATP-P2X7 axis in P2X7-overexpressing mice, which showed significantly greater stroke volumes after 24 h. In vitro experiments with primary microglia cells showed that P2X7-specific nanobodies were capable of dampening the ATP-trigged calcium-influx and formation of membrane pores measured by Fluo4 fluorescence or DAPI uptake. We found a lower caspase 1 activity and a subsequently lower IL-1b release. However, the intravenous (i.v.) injection of P2X7-specific nanobodies compared to isotype controls before the tMCAO-surgery did not result in smaller stroke size compared to isotype controls. As demonstrated by FACS, nbs had only reached brain infiltrating macrophages but not microglia. To reach microglia, we injected the P2X7-spezific nbs or the isotype directly intraventricularly (icv). 30 mg of P2X7-specific nbs proved efficient for microglial targeting, reducing post-stroke microglia activation and stroke size significantly.ConclusionHere, we demonstrate the importance of locally produced ATP for the tissue damage observed in ischemic stroke and we show the potential of icv injected P2X7-specific nbs to reduce ischemic tissue damage.

scholarly journals Caspase-8 mediates inflammation and disease in rodent malaria

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Larissa M. N. Pereira ◽  
Patrícia A. Assis ◽  
Natalia M. de Araújo ◽  
Danielle F. Durso ◽  
Caroline Junqueira ◽  
...  
Keyword(s):  
Septic Shock ◽  
Human Disease ◽  
Caspase 8 ◽  
Inflammasome Activation ◽  
Plasmodium Chabaudi ◽  
Experimental Cerebral Malaria ◽  
Rodent Malaria ◽  
Canonical Pathways ◽  
Inflammatory Caspases

Abstract Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1β and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease.

scholarly journals Mitochondrial fragmentation enables localized signaling required for cell repair

2020 ◽  
Vol 219 (5) ◽  
Author(s):  
Adam Horn ◽  
Shreya Raavicharla ◽  
Sonna Shah ◽  
Dan Cox ◽  
Jyoti K. Jaiswal
Keyword(s):  
Plasma Membrane ◽  
Calcium Influx ◽  
Cell Damage ◽  
Mitochondrial Fission ◽  
Adaptor Protein ◽  
Mitochondrial Fragmentation ◽  
Membrane Repair ◽  
Membrane Injury ◽  
Mitochondrial Signaling ◽  
Plasma Membrane Repair

Plasma membrane injury can cause lethal influx of calcium, but cells survive by mounting a polarized repair response targeted to the wound site. Mitochondrial signaling within seconds after injury enables this response. However, as mitochondria are distributed throughout the cell in an interconnected network, it is unclear how they generate a spatially restricted signal to repair the plasma membrane wound. Here we show that calcium influx and Drp1-mediated, rapid mitochondrial fission at the injury site help polarize the repair response. Fission of injury-proximal mitochondria allows for greater amplitude and duration of calcium increase in these mitochondria, allowing them to generate local redox signaling required for plasma membrane repair. Drp1 knockout cells and patient cells lacking the Drp1 adaptor protein MiD49 fail to undergo injury-triggered mitochondrial fission, preventing polarized mitochondrial calcium increase and plasma membrane repair. Although mitochondrial fission is considered to be an indicator of cell damage and death, our findings identify that mitochondrial fission generates localized signaling required for cell survival.

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