Showing papers on "Secretion published in 2017"
TL;DR: New results and concepts that are beginning to define unconventional protein secretion pathways are reviewed, which are largely triggered by stress.
399 citations
TL;DR: Current knowledge of the structure and function of T3SSs is summarized, which should aid mechanism-based drug design and facilitate their manipulation for biotechnological applications.
Abstract: Type III secretion systems (T3SSs) are protein transport nanomachines that are found in Gram-negative bacterial pathogens and symbionts. Resembling molecular syringes, T3SSs form channels that cross the bacterial envelope and the host cell membrane, which enable bacteria to inject numerous effector proteins into the host cell cytoplasm and establish trans-kingdom interactions with diverse hosts. Recent advances in cryo-electron microscopy and integrative imaging have provided unprecedented views of the architecture and structure of T3SSs. Furthermore, genetic and molecular analyses have elucidated the functions of many effectors and key regulators of T3SS assembly and secretion hierarchy, which is the sequential order by which the protein substrates are secreted. As essential virulence factors, T3SSs are attractive targets for vaccines and therapeutics. This Review summarizes our current knowledge of the structure and function of this important protein secretion machinery. A greater understanding of T3SSs should aid mechanism-based drug design and facilitate their manipulation for biotechnological applications.
398 citations
TL;DR: It is shown that MLKL-mediated NLRP3 and caspase-1 activation and the secretion of the proinflammatory cytokine IL-1β is a major determinant of necroptotic-derived inflammatory signals, and the findings suggest that NL RP3 and IL- 1β may be relevant therapeutic targets in MLK lysis-driven diseases.
Abstract: Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.
310 citations
TL;DR: Bacterial outer membrane vesicles contain immunogens but no study has yet examined their potential in treating cancer, and it is demonstrated that OMVs can suppress established tumours and prevent tumour metastasis by an interferon-γ mediated antitumor response.
Abstract: Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteolipids enriched with outer membrane proteins, to the surroundings. Outer membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used outer membrane vesicles in treating cancer thus far. Here we investigate the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show remarkable capability of bacterial outer membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects. Moreover, systematically administered bacterial outer membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such outer membrane vesicle-mediated immune response. Together, our results herein demonstrate the potential of bacterial outer membrane vesicles as effective immunotherapeutic agent that can treat various cancers without apparent adverse effects. Bacterial outer membrane vesicles (OMVs) contain immunogens but no study has yet examined their potential in treating cancer. Here, the authors demonstrate that OMVs can suppress established tumours and prevent tumour metastasis by an interferon-γ mediated antitumor response.
274 citations
TL;DR: This work shows that when intestinal epithelial cells sense an invading bacterial pathogen, they “reroute” the antimicrobial protein lysozyme through an alternative autophagy-based secretion pathway, which ensures lyso enzyme delivery to the gut lumen, which protects against further bacterial invasion.
Abstract: Intestinal Paneth cells limit bacterial invasion by secreting antimicrobial proteins, including lysozyme. However, invasive pathogens can disrupt the Golgi apparatus, interfering with secretion and compromising intestinal antimicrobial defense. Here we show that during bacterial infection, lysozyme is rerouted via secretory autophagy, an autophagy-based alternative secretion pathway. Secretory autophagy was triggered in Paneth cells by bacteria-induced endoplasmic reticulum (ER) stress, required extrinsic signals from innate lymphoid cells, and limited bacterial dissemination. Secretory autophagy was disrupted in Paneth cells of mice harboring a mutation in autophagy gene Atg16L1 that confers increased risk for Crohn’s disease in humans. Our findings identify a role for secretory autophagy in intestinal defense and suggest why Crohn’s disease is associated with genetic mutations that affect both the ER stress response and autophagy.
252 citations
TL;DR: It is shown that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL‐1β is recognized by specialized secretoryautosolic cargo receptor TRIM16 and that this receptor interacts with the R‐SNARE Sec22b to recruit cargo to the LC3‐II+ sequestration membranes.
Abstract: Autophagy is a process delivering cytoplasmic components to lysosomes for degradation. Autophagy may, however, play a role in unconventional secretion of leaderless cytosolic proteins. How secretory autophagy diverges from degradative autophagy remains unclear. Here we show that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL‐1β is recognized by specialized secretory autophagy cargo receptor TRIM16 and that this receptor interacts with the R‐SNARE Sec22b to recruit cargo to the LC3‐II + sequestration membranes. Cargo secretion is unaffected by downregulation of syntaxin 17, a SNARE promoting autophagosome–lysosome fusion and cargo degradation. Instead, Sec22b in combination with plasma membrane syntaxin 3 and syntaxin 4 as well as SNAP‐23 and SNAP‐29 completes cargo secretion. Thus, secretory autophagy utilizes a specialized cytosolic cargo receptor and a dedicated SNARE system. Other unconventionally secreted cargo, such as ferritin, is secreted via the same pathway.
229 citations
TL;DR: A single protein, HMGB1, directs the triggering of inflammation, innate and adaptive immune responses, and tissue healing after damage, and enhances the immunogenicity of mutated proteins in the tumor, promoting anti‐tumor responses and immunological memory.
Abstract: A single protein, HMGB1, directs the triggering of inflammation, innate and adaptive immune responses, and tissue healing after damage. HMGB1 is the best characterized damage-associated molecular pattern (DAMP), proteins that are normally inside the cell but are released after cell death, and allow the immune system to distinguish between antigens that are dangerous or not. Notably, cells undergoing severe stress actively secrete HMGB1 via a dedicated secretion pathway: HMGB1 is relocated from the nucleus to the cytoplasm and then to secretory lysosomes or directly to the extracellular space. Extracellular HMGB1 (either released or secreted) triggers inflammation and adaptive immunological responses by switching among multiple oxidation states, which direct the mutually exclusive choices of different binding partners and receptors. Immune cells are first recruited to the damaged tissue and then activated; thereafter, HMGB1 supports tissue repair and healing, by coordinating the switch of macrophages to a tissue-healing phenotype, activation and proliferation of stem cells, and neoangiogenesis. Inevitably, HMGB1 also orchestrates the support of stressed but illegitimate tissues: tumors. Concomitantly, HMGB1 enhances the immunogenicity of mutated proteins in the tumor (neoantigens), promoting anti-tumor responses and immunological memory. Tweaking the activities of HMGB1 in inflammation, immune responses and tissue repair could bring large rewards in the therapy of multiple medical conditions, including cancer.
217 citations
TL;DR: The data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that – despite the different vesicles biogenesis – the routes of vesicular export are adaptable.
Abstract: Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g (“microvesicles” (MV)) and 100,000 g (“exosomes”) are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane an...
208 citations
TL;DR: Links between type VI secretion, cell–cell signalling and classic siderophore receptors for iron acquisition in P. aeruginosa are revealed and are revealed to be synergistically with known iron acquisition systems.
Abstract: Iron sequestration by host proteins contributes to the defence against bacterial pathogens, which need iron for their metabolism and virulence. A Pseudomonas aeruginosa mutant lacking all three known iron acquisition systems retains the ability to grow in media containing iron chelators, suggesting the presence of additional pathways involved in iron uptake. Here we screen P. aeruginosa mutants defective in growth in iron-depleted media and find that gene PA2374, proximal to the type VI secretion system H3 (H3-T6SS), functions synergistically with known iron acquisition systems. PA2374 (which we have renamed TseF) appears to be secreted by H3-T6SS and is incorporated into outer membrane vesicles (OMVs) by directly interacting with the iron-binding Pseudomonas quinolone signal (PQS), a cell-cell signalling compound. TseF facilitates the delivery of OMV-associated iron to bacterial cells by engaging the Fe(III)-pyochelin receptor FptA and the porin OprF. Our results reveal links between type VI secretion, cell-cell signalling and classic siderophore receptors for iron acquisition in P. aeruginosa.
200 citations
TL;DR: It is reported here that ESX-1–dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores, and is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems.
Abstract: Mycobacterium tuberculosis and Mycobacterium marinum are thought to exert virulence, in part, through their ability to lyse host cell membranes. The type VII secretion system ESX-1 [6-kDa early secretory antigenic target (ESAT-6) secretion system 1] is required for both virulence and host cell membrane lysis. Both activities are attributed to the pore-forming activity of the ESX-1–secreted substrate ESAT-6 because multiple studies have reported that recombinant ESAT-6 lyses eukaryotic membranes. We too find ESX-1 of M. tuberculosis and M. marinum lyses host cell membranes. However, we find that recombinant ESAT-6 does not lyse cell membranes. The lytic activity previously attributed to ESAT-6 is due to residual detergent in the preparations. We report here that ESX-1–dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores. ESX-1–mediated lysis is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems. Our findings suggest redirection of research to understand the mechanism of ESX-1–mediated lysis.
194 citations
TL;DR: A non-metabolic function of PKM2, an enzyme associated with tumours cell reliance on aerobic glycolysis, in promoting tumour cell exosome release is revealed.
Abstract: Tumour cells secrete exosomes that are involved in the remodelling of the tumour-stromal environment and promoting malignancy. The mechanisms governing tumour exosome release, however, remain incompletely understood. Here we show that tumour cell exosomes secretion is controlled by pyruvate kinase type M2 (PKM2), which is upregulated and phosphorylated in tumours. During exosome secretion, phosphorylated PKM2 serves as a protein kinase to phosphorylate synaptosome-associated protein 23 (SNAP-23), which in turn enables the formation of the SNARE complex to allow exosomes release. Direct phosphorylation assay and mass spectrometry confirm that PKM2 phosphorylates SNAP-23 at Ser95. Ectopic expression of non-phosphorylated SNAP-23 mutant (Ser95→Ala95) significantly reduces PKM2-mediated exosomes release whereas expression of selective phosphomimetic SNAP-23 mutants (Ser95→Glu95 but not Ser20→Glu20) rescues the impaired exosomes release induced by PKM2 knockdown. Our findings reveal a non-metabolic function of PKM2, an enzyme associated with tumour cell reliance on aerobic glycolysis, in promoting tumour cell exosome release.
TL;DR: The recently discovered type IX secretion system T9SS, a complex translocon found only in some species of the Bacteroidetes phylum, remains the least explored area; however, new technical approaches and increasing scientific attention have resulted in a growing body of data.
Abstract: Protein secretion systems are vital for prokaryotic life, as they enable bacteria to acquire nutrients, communicate with other species, defend against biological and chemical agents, and facilitate disease through the delivery of virulence factors In this review, we will focus on the recently discovered type IX secretion system (T9SS), a complex translocon found only in some species of the Bacteroidetes phylum T9SS plays two roles, depending on the lifestyle of the bacteria It provides either a means of movement (called gliding motility) for peace-loving environmental bacteria or a weapon for pathogens The best-studied members of these two groups are Flavobacterium johnsoniae, a commensal microorganism often found in water and soil, and Porphyromonas gingivalis, a human oral pathogen that is a major causative agent of periodontitis In P gingivalis and some other periodontopathogens, T9SS translocates proteins, especially virulence factors, across the outer membrane (OM) Proteins destined for secretion bear a conserved C-terminal domain (CTD) that directs the cargo to the OM translocon At least 18 proteins are involved in this still enigmatic process, with some engaged in the post-translational modification of T9SS cargo proteins Upon translocation across the OM, the CTD is removed by a protease with sortase-like activity and an anionic LPS is attached to the newly formed C-terminus As a result, a cargo protein could be secreted into the extracellular milieu or covalently attached to the bacterial surface T9SS is regulated by a two-component system; however, the precise environmental signal that triggers it has not been identified Exploring unknown systems contributing to bacterial virulence is exciting, as it may eventually lead to new therapeutic strategies During the past decade, the major components of T9SS were identified, as well as hints suggesting the possible mechanism of action In addition, the list of characterized cargo proteins is constantly growing The actual structure of the translocon, situated in the OM of bacteria, remains the least explored area; however, new technical approaches and increasing scientific attention have resulted in a growing body of data Therefore, we present a compact up-to-date review of this topic
TL;DR: The major facilitator superfamily transporter 2b (Mfsd2b), an orphan transporter, is essential for S1P export from red blood cells and platelets and is critical for red blood cell morphology, revealing an unexpected physiological role ofred blood cells in sphingolipid metabolism in circulation.
Abstract: Sphingosine-1-phosphate (S1P), a potent signalling lipid secreted by red blood cells and platelets, plays numerous biologically significant roles. However, the identity of its long-sought exporter is enigmatic. Here we show that the major facilitator superfamily transporter 2b (Mfsd2b), an orphan transporter, is essential for S1P export from red blood cells and platelets. Comprehensive lipidomic analysis indicates a dramatic and specific accumulation of S1P species in Mfsd2b knockout red blood cells and platelets compared with that of wild-type controls. Consistently, biochemical assays from knockout red blood cells, platelets, and cell lines overexpressing human and mouse Mfsd2b proteins demonstrate that Mfsd2b actively exports S1P. Plasma S1P level in knockout mice is significantly reduced by 42-54% of that of wild-type level, indicating that Mfsd2b pathway contributes approximately half of the plasma S1P pool. The reduction of plasma S1P in knockout mice is insufficient to cause blood vessel leakiness, but it does render the mice more sensitive to anaphylactic shock. Stress-induced erythropoiesis significantly increased plasma S1P levels and knockout mice were sensitive to these treatments. Surprisingly, knockout mice exhibited haemolysis associated with red blood cell stomatocytes, and the haemolytic phenotype was severely increased with signs of membrane fragility under stress erythropoiesis. We show that S1P secretion by Mfsd2b is critical for red blood cell morphology. Our data reveal an unexpected physiological role of red blood cells in sphingolipid metabolism in circulation. These findings open new avenues for investigating the signalling roles of S1P derived from red blood cells and platelets.
TL;DR: Evidence is presented that ESX‐1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis, and identifies an as yet unknown function for DIM in the infection process.
Abstract: Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT-6). This small protein, which is secreted by the type VII secretion system ESX-1 (T7SS/ESX-1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock-out and/or knock-in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX-1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to the host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb.
TL;DR: The demonstration that EV‐YF1 confers cardioprotection highlights the potential importance of diverse exosomal contents of unknown function, above and beyond the usual suspects (e.g., microRNAs and proteins).
Abstract: Cardiosphere‐derived cells (CDCs) reduce myocardial infarct size via secreted extracellular vesicles (CDC‐EVs), including exosomes, which alter macrophage polarization. We questioned whether short non‐coding RNA species of unknown function within CDC‐EVs contribute to cardioprotection. The most abundant RNA species in CDC‐EVs is a Y RNA fragment (EV‐YF1); its relative abundance in CDC‐EVs correlates with CDC potency in vivo . Fluorescently labeled EV‐YF1 is actively transferred from CDCs to target macrophages via CDC‐EVs. Direct transfection of macrophages with EV‐YF1 induced transcription and secretion of IL‐10. When cocultured with rat cardiomyocytes, EV‐YF1‐primed macrophages were potently cytoprotective toward oxidatively stressed cardiomyocytes through induction of IL‐10. In vivo, intracoronary injection of EV‐YF1 following ischemia/reperfusion reduced infarct size. A fragment of Y RNA, highly enriched in CDC‐EVs, alters Il10 gene expression and enhances IL‐10 protein secretion. The demonstration that EV‐YF1 confers cardioprotection highlights the potential importance of diverse exosomal contents of unknown function, above and beyond the usual suspects (e.g., microRNAs and proteins).
TL;DR: A mechanism through which TMEM24, a lipid transport protein that concentrates at endoplasmic reticulum–plasma membrane contact sites, regulates the pulsatility of cytosolic Ca2+ and phosphoinositide signaling is reported, which regulates pulsatile insulin secretion during the slow insulin release phase.
Abstract: INTRODUCTION Insulin is secreted by pancreatic β cells in response to glucose stimulation. Its release is controlled by the interplay of calcium and phosphoinositide signaling pathways. A rapid release phase, in which insulin containing granules that are already docked and primed at the plasma membrane (PM) undergo exocytosis, is followed by slow release. In this second phase, granules are docked and primed and then released in a series of bursts, each triggered by a spike in cytosolic Ca 2+ . RATIONALE To better understand the molecular basis underlying insulin secretion, we characterized TMEM24, a protein enriched in neuroendocrine cells previously suggested to be required for a normal secretory response. RESULTS We found that TMEM24 is an endoplasmic reticulum (ER) protein that concentrates at ER-PM contact sites, where it tethers the two bilayers. TMEM24 binding “in trans” to the PM is negatively regulated by phosphorylation in response to elevation of cytosolic Ca 2+ , so that TMEM24 transiently dissociates from the PM as Ca 2+ concentration spikes and then reassociates with this membrane upon dephosphorylation. Additionally, TMEM24 contains a lipid transport module of the synaptotagmin-like, mitochondrial and lipid-binding protein (SMP) family, which we structurally characterized and showed to bind glycerolipids with a preference for phosphatidylinositol (PI). Thus, TMEM24 helps deliver PI, which is synthesized in the ER, to the PM, where it is converted to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ] to replenish pools of this lipid hydrolyzed during glucose-stimulated signaling. Supporting a key role of TMEM24 in the coordination of Ca 2+ and phosphoinositide signaling, the lipid transport function of TMEM24 is essential for sustaining the intracellular Ca 2+ oscillations that trigger bursts of insulin granule release and hence insulin secretion. PI(4,5)P 2 is required for Ca 2+ -dependent exocytosis. It also controls the activity of PM ion channels that regulate cytosolic Ca 2+ levels and is the precursor of IP 3 , which also helps to modulate cytosolic Ca 2+ by triggering Ca 2+ release from the ER. Thus, in insulin-secreting cells, TMEM24 participates in coordinating Ca 2+ and phosphoinositide signaling pathways to cause pulsatile insulin secretion (see the figure). CONCLUSIONS Our findings implicate ER-PM contact sites and an ER resident lipid-transfer protein in the direct regulation of PM phosphoinositide pools, offering fresh insights into the mechanisms of cellular phosphoinositide dynamics. More specifically, they elaborate the mechanisms underlying insulin secretion, which is impaired in patients with type II diabetes and may ultimately have therapeutic ramifications.
11 Apr 2017
TL;DR: Study of the role of CagA during neoplastic transformation will provide an excellent opportunity to understand molecular processes underlying “Hit-and-Run” carcinogenesis.
Abstract: Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor of gastric cancer. The cagA gene-encoded CagA protein is delivered into gastric epithelial cells via bacterial type IV secretion, where it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Delivered CagA then acts as a non-physiological scaffold/hub protein by interacting with multiple host signaling molecules, most notably the pro-oncogenic phosphatase SHP2 and the polarity-regulating kinase PAR1/MARK, in both tyrosine phosphorylation-dependent and -independent manners. CagA-mediated manipulation of intracellular signaling promotes neoplastic transformation of gastric epithelial cells. Transgenic expression of CagA in experimental animals has confirmed the oncogenic potential of the bacterial protein. Structural polymorphism of CagA influences its scaffold function, which may underlie the geographic difference in the incidence of gastric cancer. Since CagA is no longer required for the maintenance of established gastric cancer cells, studying the role of CagA during neoplastic transformation will provide an excellent opportunity to understand molecular processes underlying "Hit-and-Run" carcinogenesis.
TL;DR: It is found that a single administration of EVs improved functional recovery, fiber tract integrity, axonal sprouting and white matter repair markers in an experimental animal model of subcortical stroke and presents a new perspective in the development of biologics for brain repair.
Abstract: Mesenchymal stem cells have previously been shown to mediate brain repair after stroke; they secrete 50–100 nm complexes called extracellular vesicles (EVs), which could be responsible for provoking neurovascular repair and functional recovery. EVs have been observed by electron microscopy and NanoSight, and they contain associated proteins such as CD81 and Alix. This purified, homogeneous population of EVs was administered intravenously after subcortical stroke in rats. To evaluate the EVs effects, we studied the biodistribution, proteomics analysis, functional evaluation, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers. We found that a single administration of EVs improved functional recovery, fiber tract integrity, axonal sprouting and white matter repair markers in an experimental animal model of subcortical stroke. EVs were found in the animals’ brain and peripheral organs after euthanasia. White matter integrity was in part restored by EVs administration mediated by molecular repair factors implicated in axonal sprouting, tract connectivity, remyelination and oligodendrogenesis. These findings are associated with improved functional recovery. This novel role for EVs presents a new perspective in the development of biologics for brain repair.
TL;DR: A role of LMP1 is described in EV production that requires CD63 and an extensive demonstration of CD63-mediated exosomal LMP2 release that is distinct from lipid raft trafficking is provided, suggesting that CD63 is a critical player in L MP1Exosomal trafficking and LMP 1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP3 signaling.
Abstract: Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-κB and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis.
TL;DR: Manumycin-A (MA), a natural microbial metabolite, was identified as an inhibitor of exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) C4-2B, but not the normal RWPE-1, cells.
TL;DR: Mechanistic insights are provided into how chemotherapy triggers innate immune responses causing intestinal toxicity, and new chemotherapy regimens are revealed that maintain anti-tumour effects but circumvent the associated adverse inflammatory response.
Abstract: Chemotherapies are known often to induce severe gastrointestinal tract toxicity but the underlying mechanism remains unclear. This study considers the widely applied cytotoxic agent irinotecan (CPT-11) as a representative agent and demonstrates that treatment induces massive release of double-strand DNA from the intestine that accounts for the dose-limiting intestinal toxicity of the compound. Specifically, "self-DNA" released through exosome secretion enters the cytosol of innate immune cells and activates the AIM2 (absent in melanoma 2) inflammasome. This leads to mature IL-1β and IL-18 secretion and induces intestinal mucositis and late-onset diarrhoea. Interestingly, abrogation of AIM2 signalling, either in AIM2-deficient mice or by a pharmacological inhibitor such as thalidomide, significantly reduces the incidence of drug-induced diarrhoea without affecting the anticancer efficacy of CPT-11. These findings provide mechanistic insights into how chemotherapy triggers innate immune responses causing intestinal toxicity, and reveal new chemotherapy regimens that maintain anti-tumour effects but circumvent the associated adverse inflammatory response.
TL;DR: It is proposed that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D.
Abstract: The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D.
TL;DR: This work discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9, which provides reliable targets for both highly specific diagnosis and immune therapy of AML.
TL;DR: It is demonstrated that plasma GLP-1 levels are rapidly increased by lipopolysaccharide (LPS) administration in mice via a Toll-like receptor 4 (TLR4)-dependent mechanism, linking glucagon-like peptide secretion to gut inflammation.
TL;DR: Genome-wide RNAi screening revealed numerous host factors involved in H. pylori-, but not IL-1β- and TNF-α-dependent NF-κB regulation, highlighting the ALPK1-TIFA axis as a core innate immune pathway.
TL;DR: The intracellular pathogenic bacterium Edwardsiella tarda was demonstrated to activate NLRC4 andNLRP3 inflammasomes via a type III secretion system (T3SS), and to inhibit NLRP3inflammasome via a types VI and T6SS secretion system, indicating the antagonistic roles of these systems in inflammaome signaling.
TL;DR: The molecular basis of the extracellular function of ISG15 and the initial outside-in signaling events that driveISG15-dependent cytokine secretion are established.
TL;DR: The results of the present study suggest that the mechanosensitive ion channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransduction.
Abstract: Key points
The gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransduction is unknown.
In the present study, we show, for the first time, that the mechanosensitive ion channel Piezo2 is specifically expressed by the human and mouse EC cells.
Activation of Piezo2 by mechanical forces results in a characteristic ionic current, the release of serotonin and stimulation of gastrointestinal secretion.
Piezo2 inhibition by drugs or molecular knockdown decreases mechanosensitive currents, serotonin release and downstream physiological effects.
The results of the present study suggest that the mechanosensitive ion channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransduction.
Abstract
The enterochromaffin (EC) cell in the gastrointestinal (GI) epithelium is the source of nearly all systemic serotonin (5-hydroxytryptamine; 5-HT), which is an important neurotransmitter and endocrine, autocrine and paracrine hormone. The EC cell is a specialized mechanosensor, and it is well known that it releases 5-HT in response to mechanical forces. However, the EC cell mechanotransduction mechanism is unknown. The present study aimed to determine whether Piezo2 is involved in EC cell mechanosensation. Piezo2 mRNA was expressed in human jejunum and mouse mucosa from all segments of the small bowel. Piezo2 immunoreactivity localized specifically within EC cells of human and mouse small bowel epithelium. The EC cell model released 5-HT in response to stretch, and had Piezo2 mRNA and protein, as well as a mechanically-sensitive inward non-selective cation current characteristic of Piezo2. Both inward currents and 5-HT release were inhibited by Piezo2 small interfering RNA and antagonists (Gd3+ and D-GsMTx4). Jejunum mucosal pressure increased 5-HT release and short-circuit current via submucosal 5-HT3 and 5-HT4 receptors. Pressure-induced secretion was inhibited by the mechanosensitive ion channel antagonists gadolinium, ruthenium red and D-GsMTx4. We conclude that the EC cells in the human and mouse small bowel GI epithelium selectively express the mechanosensitive ion channel Piezo2, and also that activation of Piezo2 by force leads to inward currents, 5-HT release and an increase in mucosal secretion. Therefore, Piezo2 is critical to EC cell mechanosensitivity and downstream physiological effects.
TL;DR: The host can detect the presence of bacterial protein secretion systems as hallmarks of PVs, and insertion of bacterial secretion systems into PV membranes stimulates Galectin-3–dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program.
Abstract: Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as "patterns of pathogenesis" associated with PVs. We report that the delivery of GBP2 to Legionella-containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia-containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella-containing vacuoles or YCVs is substantially diminished in Galectin-3-deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3-dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program.
TL;DR: Recent studies that identified the roles of the inflammasome, inflammatory caspases, and GSDMD in pyroptosis are reviewed and some of the outstanding questions about their roles in innate immunity, control of infection, and sepsis are highlighted.
Abstract: Immune cells and skin and mucosal epithelial cells recognize invasive microbes and other signs of danger to sound alarms that recruit responder cells and initiate an immediate "innate" immune response. An especially powerful alarm is triggered by cytosolic sensors of invasive infection that assemble into multimolecular complexes, called inflammasomes, that activate the inflammatory caspases, leading to maturation and secretion of proinflammatory cytokines and pyroptosis, an inflammatory death of the infected cell. Work in the past year has defined the molecular basis of pyroptosis. Activated inflammatory caspases cleave Gasdermin D (GSDMD), a cytosolic protein in immune antigen-presenting cells and epithelia. Cleavage separates the autoinhibitory C-terminal fragment from the active N-terminal fragment, which moves to the cell membrane, binds to lipids on the inside of the cell membrane, and oligomerizes to form membrane pores that disrupt cell membrane integrity, causing death and leakage of small molecules, including the proinflammatory cytokines and GSDMD itself. GSDMD also binds to cardiolipin on bacterial membranes and kills the very bacteria that activate the inflammasome. GSDMD belongs to a family of poorly studied gasdermins, expressed in the skin and mucosa, which can also form membrane pores. Spontaneous mutations that disrupt the binding of the N- and C-terminal domains of other gasdermins are associated with alopecia and asthma. Here, we review recent studies that identified the roles of the inflammasome, inflammatory caspases, and GSDMD in pyroptosis and highlight some of the outstanding questions about their roles in innate immunity, control of infection, and sepsis.