Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of ...bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.
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▸ XIAP is required for NOD2-dependent signaling and inflammation in vivo ▸ XIAP ubiquitylates RIPK2 after NOD2 stimulation to facilitate LUBAC recruitment ▸ LUBAC regulates NOD2-dependent immune signaling ▸ XLP-2-derived XIAP mutations impair ubiquitin ligase activity and NOD2 signaling
The E3 ubiquitin ligase Parkin is a key effector of the removal of damaged mitochondria by mitophagy. Parkin determines cell fate in response to mitochondrial damage, with its loss promoting early ...onset Parkinson's disease and potentially also cancer progression. Controlling a cell's apoptotic response is essential to co‐ordinate the removal of damaged mitochondria. We report that following mitochondrial damage‐induced mitophagy, Parkin directly ubiquitinates the apoptotic effector protein BAK at a conserved lysine in its hydrophobic groove, a region that is crucial for BAK activation by BH3‐only proteins and its homo‐dimerisation during apoptosis. Ubiquitination inhibited BAK activity by impairing its activation and the formation of lethal BAK oligomers. Parkin also suppresses BAX‐mediated apoptosis, but in the absence of BAX ubiquitination suggesting an indirect mechanism. In addition, we find that BAK‐dependent mitochondrial outer membrane permeabilisation during apoptosis promotes PINK1‐dependent Parkin activation. Hence, we propose that Parkin directly inhibits BAK to suppress errant apoptosis, thereby allowing the effective clearance of damaged mitochondria, but also promotes clearance of apoptotic mitochondria to limit their potential pro‐inflammatory effect.
Synopsis
The ubiquitin ligase Parkin plays a protective role in neurodegenerative disease by removing damaged mitochondria and preventing apoptosis, and by limiting the functions of pro‐apoptotic effector proteins BAK and BAX. Defective control of apoptosis may contribute to the pathogenesis of early onset Parkinson's Disease caused by Parkin mutations.
Parkin activity is induced by mitochondrial damage during apoptosis.
Parkin mono‐ and di‐ubiquitinates BAK at a conserved lysine in its hydrophobic groove.
Ubiquitination reduces BAK oligomerisation and apoptotic activity on mitochondria.
Parkin prevents BAX mitochondrial localisation and apoptotic activity independent of ubiquitination.
Certain Parkinson's Disease‐associated Parkin mutants cannot ubiquitinate BAK and restrain it on mitochondria.
Autophagic clearance of damaged mitochondria and suppression of apoptotic effector proteins are coordinated functions of the Parkin ubiquitin ligase that are lost upon Parkinson's Disease‐associated mutations.
Mixed lineage kinase domain‐like (MLKL) is the executioner in the caspase‐independent form of programmed cell death called necroptosis. Receptor‐interacting serine/threonine protein kinase 3 (RIPK3) ...phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis‐specific multi‐mono‐ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin‐insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane‐located deubiquitylating enzyme. Oligomerization‐induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome‐ and lysosome‐dependent turnover. Using a MLKL‐DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto‐activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL‐induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.
SYNOPSIS
RIPK3 phosphorylates the necroptotic effector molecule MLKL leading to its oligomerization and translocation to the plasma membrane to kill cells. MLKL becomes multi mono‐ubiquitylated in an oligomerization dependent manner. Forced de‐ubiquitylation of MLKL increases MLKL's cytotoxic potential and confers RIPK3 and necroptotic stimulus independent activation and cell death.
UbiCRest analysis shows that MLKL becomes multi mono‐ubiquitylated contemporaneously with activating phosphorylation and translocation to membranes.
Analysis of gain and loss of function MLKL mutants indicates that MLKL oligomerization is required for necroptosis induced ubiquitylation.
MLKL‐deubiquitylating enzyme (DUB) fusions kill cells more rapidly than MLKL‐catalytically dead DUB fusions and can induce necroptosis without a necroptotic stimulus, suggesting that ubiquitylation serves as a brake on MLKL's cytotoxic potential.
Mono‐ubiquitylation of the necroptotic effector molecule MLKL promotes its proteasome‐ and lysosome‐mediated turnover to restrains its necroptosis‐inducing activity.
Loss of inhibitor of apoptosis proteins (IAPs), particularly cIAP1, can promote production of tumor necrosis factor (TNF) and sensitize cancer cell lines to TNF-induced necroptosis by promoting ...formation of a death-inducing signaling complex containing receptor-interacting serine/threonine-protein kinase (RIPK) 1 and 3. To define the role of IAPs in myelopoiesis, we generated a mouse with cIAP1, cIAP2, and XIAP deleted in the myeloid lineage. Loss of cIAPs and XIAP in the myeloid lineage caused overproduction of many proinflammatory cytokines, resulting in granulocytosis and severe sterile inflammation. In vitro differentiation of macrophages from bone marrow in the absence of cIAPs and XIAP led to detectable levels of TNF and resulted in reduced numbers of mature macrophages. The cytokine production and consequent cell death caused by IAP depletion was attenuated by loss or inhibition of TNF or TNF receptor 1. The loss of RIPK1 or RIPK3, but not the RIPK3 substrate mixed lineage kinase domain-like protein, attenuated TNF secretion and thereby prevented apoptotic cell death and not necrosis. Our results demonstrate that cIAPs and XIAP together restrain RIPK1- and RIPK3-dependent cytokine production in myeloid cells to critically regulate myeloid homeostasis.
•cIAPs and XIAP negatively regulate cytokine production, including TNF to disrupt myeloid lineage differentiation.•IAPs prevent RIPK1 and RIPK3 activity to limit cytokine production prior to cell death.
Inhibitor of apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP (X‐linked IAP) regulate apoptosis and cytokine receptor signalling, but their overlapping functions make it difficult to distinguish their ...individual roles. To do so, we deleted the genes for IAPs separately and in combination. While lack of any one of the IAPs produced no overt phenotype in mice, deletion of cIap1 with cIap2 or Xiap resulted in mid‐embryonic lethality. In contrast, Xiap−/−cIap2−/− mice were viable. The death of cIap2−/−cIap1−/− double mutants was rescued to birth by deletion of tumour necrosis factor (TNF) receptor 1, but not TNFR2 genes. Remarkably, hemizygosity for receptor‐interacting protein kinase 1 (Ripk1) allowed Xiap−/−cIap1−/− double mutants to survive past birth, and prolonged cIap2−/−cIap1−/− embryonic survival. Similarly, deletion of Ripk3 was able to rescue the mid‐gestation defect of cIap2−/−cIap1−/− embryos, as these embryos survived to E15.5. cIAPs are therefore required during development to limit activity of RIP kinases in the TNF receptor 1 signalling pathway.
The inhibitor of apoptosis proteins cIAP1, cIAP2, and XIAP exert overlapping functions in apoptosis and cytokine signalling. A series of single‐ and double‐knockout mice reveal an essential function of IAP proteins in preventing TNF receptor 1‐induced, RIP kinase 1‐ and 3‐dependent cell death during embryogenesis.
Inhibitors of apoptosis (IAPs) proteins are critical regulators of innate immune signaling pathways and therefore have potential as drug targets. X-linked IAP (XIAP) and cellular IAP1 and IAP2 (cIAP1 ...and cIAP2) are E3 ligases that have been shown to be required for signaling downstream of NOD2, an intracellular receptor for bacterial peptidoglycan. We used genetic and biochemical approaches to compare the responses of IAP-deficient mice and cells to NOD2 stimulation. In all cell types tested, XIAP is the only IAP required for signaling immediately downstream of NOD2, while cIAP1 and cIAP2 are dispensable for NOD2-induced nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. However, mice lacking cIAP1 or TNFR1 have a blunted cytokine response to NOD2 stimulation. We conclude that cIAPs regulate NOD2-dependent autocrine TNF signaling in vivo and highlight the importance of physiological context in the interplay of innate immune signaling pathways.
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•XIAP is the only IAP required for NF-κB and MAPK activation downstream of NOD2•NOD2-driven cytokine production relies on a TNF-dependent amplification loop•Dendritic-like cells are the first responders to MDP stimulation in vivo
Stafford et al. show that XIAP is the only IAP required for the initial RIPK2/NOD2-dependent response to MDP, while autocrine TNF is required to amplify cytokine production in a cIAP1-dependent manner.
Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, ...Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Progranulin (proepithelin, granulin precursor) has been recently suggested to exhibit anti‐inflammatory properties by directly binding to tumour necrosis factor (TNF) receptors and thereby inhibiting ...TNF signalling by Tang et al. This finding was challenged by Chen et al. and no interaction between progranulin and TNF receptor (TNFR) 1 or 2 was observed. We tested the ability of recombinant progranulin from different commercial sources to inhibit TNF‐ or lymphotoxin‐α‐induced signalling through TNFR1. We observed that progranulin does not affect signalling and cell death induction downstream of TNF or lymphotoxin‐α. Our results suggest that the anti‐inflammatory role of progranulin is not mediated through direct inhibition of TNFR1.
Neutrophils have been shown to contribute to the pathophysiology of hidradenitis suppurativa (HS), a chronic, painful and debilitating inflammatory skin disease, yet their exact role remains to be ...fully defined. Granulocyte colony-stimulating factor (G-CSF), a major regulator of neutrophil development and survival, can be blocked by the novel, fully human anti-G-CSF receptor (G-CSFR) monoclonal antibody CSL324.
We investigated the activation and migration of neutrophils in HS and the impact of blocking G-CSFR with CSL324.
Biopsy and peripheral blood samples were taken from participants of two studies: 2018.206, a noninterventional research study of systemic and dermal neutrophils and inflammatory markers in patients with neutrophilic skin diseases, and CSL324_1001 (ACTRN12616000846426), a single-dose ascending and repeated dose, randomized, double-blind, placebo-controlled study to assess the safety, pharmacokinetics and pharmacodynamics of CSL324 in healthy adult subjects. Ex vivo experiments were performed, including neutrophil enumeration and immunophenotyping, migration, receptor occupancy and transcriptome analysis.
The number of cells positive for the neutrophil markers myeloperoxidase (MPO) and neutrophil elastase (NE) was significantly higher in HS lesions compared with biopsies from healthy donors (HDs) (P < 0.0001 and P = 0.0223, respectively). In peripheral blood samples, mean neutrophil counts were significantly higher in patients with HS than in HDs (2.98 vs. 1.60 × 109 L-1, respectively; P = 8.8 × 10-4). Neutrophil migration pathways in peripheral blood were increased in patients with HS and their neutrophils demonstrated an increased migration phenotype, with higher mean CXCR1 on the surface of neutrophils in patients with HS (24453.20 vs. 20798.47 for HD; P = 0.03). G-CSF was a key driver of the transcriptomic changes in the peripheral blood of patients with HS and was elevated in serum from patients with HS compared with HDs (mean 6.61 vs. 3.84 pg mL-1, respectively; P = 0.013). Administration of CSL324 inhibited G-CSF-induced transcriptional changes in HDs, similar to those observed in the HS cohort, as highlighted by expression changes in genes related to neutrophil migratory capacity.
Data suggest that neutrophils contribute to HS pathophysiology and that neutrophils are increased in lesions due to an increase in G-CSF-driven migration. CSL324 counteracted G-CSF-induced transcriptomic changes and blocked neutrophil migration by reducing cell-surface levels of chemokine receptors.
Progranulin (proepithelin, granulin precursor) has been recently suggested to exhibit anti-inflammatory properties by directly binding to tumour necrosis factor (TNF) receptors and thereby inhibiting ...TNF signalling by Tang et al. This finding was challenged by Chen et al. and no interaction between progranulin and TNF receptor (TNFR) 1 or 2 was observed. We tested the ability of recombinant progranulin from different commercial sources to inhibit TNF- or lymphotoxin-α-induced signalling through TNFR1. We observed that progranulin does not affect signalling and cell death induction downstream of TNF or lymphotoxin-α. Our results suggest that the anti-inflammatory role of progranulin is not mediated through direct inhibition of TNFR1.
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