Haploid genetic screen for components of MLKL-mediated cell death
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ABSTRACT:
PROVIDER: PRJEB26694 | ENA |
REPOSITORIES: ENA
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Project description:One hundred million HAP1 Tet-On-MLKLTE/SD cells were mutagenized with the GFP gene trap retrovirus, as described (PMID: 21623355). The selection experiment was performed by inducing MLKLTE/SD expression with 200 ng/mL DOX over five days and subsequently allowing resistant colonies to grow for four days after withdrawing DOX. Resistant colonies were then pooled together and genomic DNA was extracted from the pool (Qiagen, 51306). Retroviral gene trap insertion sites were amplified using a linear amplification (LAM) PCR protocol as described previously (PMID: 21623355), sequenced using the Genome Analyzer (Illumina).
2018-05-16 | E-MTAB-6777 | biostudies-arrayexpress
Project description:The activation of Mixed Lineage Kinase-Like (MLKL) by Receptor Interacting Protein Kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here we show that during necroptosis, MLKL-dependent calcium (Ca++) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM “bubbles” with exposed PS that are released from the surface of the otherwise intact cell. Components of the ESCRT machinery are required for formation of these bubbles, and act to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT, cells undergoing necroptosis can express chemokines and other regulatory molecules, and promote antigenic cross-priming of CD8+ T cells.
2017-06-16 | GSE85660 | GEO
Project description:Caspase-8 is a protease with both pro-death and pro-survival functions: it is required for apoptosis induced by death receptors such as TNFR1 (tumour necrosis factor receptor 1), and it has a critical role in suppressing necroptosis mediated by the kinase RIPK3 (receptor interacting protein kinase 3) and the pseudokinase MLKL (mixed lineage kinase-like). Mice lacking caspase-8 display MLKL-dependent embryonic lethality, as do mice expressing catalytically inactive caspase-8 mutant C362A. However, Casp8C362A/C362A Mlkl-/- mice die in the perinatal period, whereas Casp8-/- Mlkl-/- mice are viable, indicating that inactive caspase-8 also has a pro-death scaffolding function. Here we show that caspase-8 C362A triggers ASC speck formation and caspase-1-dependent pyroptosis in MLKL-deficient intestinal epithelial cells (IECs) around embryonic day 18. Pyroptosis contributed to the perinatal lethal phenotype because a number of Casp8 C362A/C362A Mlkl-/- Casp1-/- mice survived beyond weaning. Transfection studies suggested inactive caspase-8 adopts a distinct conformation to wild-type caspase-8, enabling it to engage the caspase-1 adaptor ASC. Wild-type caspase-8 was found in the Triton X-100 soluble fraction, whereas wild-type caspase-8 inhibited with the pan-caspase inhibitor emricasan, or inactive caspase-8 mutant C362A, were detected in the insoluble fraction. Moreover, inhibited or inactive caspase-8 shifted ASC into the insoluble fraction. Perinatal lethality was recapitulated when expression of caspase-8 C362A was restricted to IECs, but intriguingly, only in the absence of MLKL. Hence, unanticipated plasticity in death pathways is revealed such that IECs can undergo caspase-1-dependent death when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.
2019-09-20 | GSE132133 | GEO
Project description:We undertook a unbiased genome-wide haploid genetic screen to identify new components in interferon lambda signaling. In addition, we performed a genome-wide screen to identify genes that repress spontaneous activation of interferon stimulated genes in the absence of interferon. Both of these screens were performed using a HAP1 cell line containing GFP reporter under the transcriptional regulation of the Interferon-Stimulated Response Element from IFIT2. We also overexpressed IL28RA (IFNLR1) in this cell line, in order to sensitize the cells to type III interferon
2017-07-25 | E-MTAB-5866 | biostudies-arrayexpress
Project description:Caspase-8 is a protease with both pro-death and pro-survival functions: it is required for apoptosis induced by death receptors such as TNFR1 (tumor necrosis factor receptor 1) 1, and it has a critical role in suppressing necroptosis mediated by the kinase RIPK3 (receptor interacting protein kinase 3) and the pseudokinase MLKL (mixed lineage kinase-like) 2-4. Mice lacking caspase-8 display MLKL-dependent embryonic lethality 4, as do mice expressing catalytically inactive caspase-8 mutant C362A. However, Casp8C362A/C362A Mlkl-/- mice die in the perinatal period, whereas Casp8-/- Mlkl-/- mice are viable 4, indicating that inactive caspase-8 also has a pro-death scaffolding function. Here we show that inactive caspase-8 activates pyroptosis in MLKL-deficient intestinal epithelial cells around embryonic day 18, triggering the formation of ASC specks. Accordingly, intestinal atrophy and perinatal lethality in Casp8C362A/C362A Mlkl-/- mice was prevented by loss of caspase-1. In transfection studies, inactive caspase-8 mutants C362A or C362S were found in both the triton X-100 insoluble and soluble fractions, whereas wild-type caspase-8 existed only in the soluble fraction. Moreover, inactive caspase-8 shifted co-transfected ASC into the insoluble fraction, whereas wild-type caspase-8 did not. Thus, a defense mechanism is revealed that would allow intestinal epithelial cell death in the face of pathogens expressing virulence factors to inhibit caspase-8-dependent apoptosis and necroptosis.
2019-09-20 | GSE125625 | GEO
Project description:Neuroinflammatory processes are a prominent contributor to the pathology of Parkinson’s disease (PD), characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and deposits of α-synuclein aggregates. MLKL-mediated cell necroptosis might occur in the onset of PD and lead to neuronal dopaminergic degeneration. However, the link between α-synuclein, neuroinflammatory processes, and neurodegeneration in PD remains unclear. Here, our in vitro study indicated that inhibition of MLKL exerted a protective effect against 6-OHDA- and TNF-α-induced neuronal cell death. Furthermore, we created a mouse model (Tg-Mlkl-/-) with typical progressive Parkinson traits by crossbreeding SNCA A53T transgenic mice with MLKL knockout mice. Tg-Mlkl-/ mice displayed dramatically improved motor symptoms and reduced hyperphosphorylated α-synuclein expression. More data suggested that MLKL deficiency protected dopaminergic neurons, blocked neuronal cell death, and attenuated neuroinflammation by inhibiting the activation of the microglia and astrocytes. Single-cell RNA-seq analysis revealed reduced microglial cells and damped neuron death in the SN of the Tg-Mlkl-/- mice. Subcluster analysis identified a unique cell type-specific transcriptome profiling in the MLKL deficiency mice. Thus, MLKL represents a critical therapeutic target for reducing neuroinflammation and preventing dopaminergic neuron degeneration.
2023-12-14 | GSE197679 | GEO
Project description:Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase 8-independent form of programmed cell death called necroptosis. Once Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) is activated by upstream cell death signals, it phosphorylates MLKL and triggers the oligomerization and membrane translocation required for MLKL induced membrane disruption. Besides phosphorylation, MLKL also undergoes ubiquitylation during the early stages of 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 on biological membranes, and requires its activation and oligomerisation. Inactive MLKL mutants recruited to membranes during necroptosis are ubiquitylated but this results in their proteasome and lysosome dependent turnover. We identified several ubiquitylated lysines however mutation of these did not affect MLKL ubiquitylation in response to a necroptotic stimulus.
2021-04-28 | PXD015537 | Pride
Project description:Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune disease and IL-1R-dependent caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1R-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigated the mechanisms controlling IL-1α/β release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1/Ripk3/Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38-dependent Ripk1-independent IL-1 and TNF production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 MAP kinase activation to control TNF and IL-1α/β transcription, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3/Mlkl-dependent cell death and concomitant IL-1α/β release.
2019-11-12 | GSE139223 | GEO
Project description:PIP2 enhances MLKL channel activity in a direct interaction manner and this gain of function promotes both necroptosis and inflammation. Previous studies have reported that phospholipids assisted MLKL recruitment and translocation which facilitates its mediated function like liposome leaking. Here, our result support MLKL act as ion channel and is finely tuned by PIP2. In the immune process especially, PIP2, as an important modulator, promotes intracellular potassium depletion and trigger inflammation which mediated bythrough MLKL channel function. Defining the role of MLKL channel function in MLKL-induced necroptosis or other potential necroptotic models will extend our understanding of the programmed cell death and critically inform the development and testing of new disease-specific, Anti-inflammatory, therapeutic strategies.
2022-11-05 | PXD030814 | Pride
Project description:Purpose: Necroptosis as been implicated in various deseases. The goal of this study is to invastigate the impact of RIPK3 and MLKL in the lipid metabolism of adipocytes. Methods: 3T3-L1 preadipocytes invalidated or not for RIPK3 or MLKL were exposed differenciated into mature adipocytes and the mRNA profiles of wild type (WT), RIPK3-/- knockout (RIPK3-KO) or MLKL-/- knockout 3T3-L1 cells control (J0) or differenciated into mature adipocytes (J7) were generated by deep sequencing, in 3 copies, using Illumina NOVAseq 6000 plateform. Differential expression analysis between two conditions/groups (five biological replicates per condition) was performed using DESeq2 R package. Genes with an adjusted P value < 0.05 found by DESeq2 were assigned as differentially expressed. qRT–PCR validation was performed using SYBR Green assays Results: The DEGs were clustered using a hierarchical clustering algorithm, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis unveiled a clear reduction in the expression of genes involved in the early or late stages of adipogenesis in MLKL-KO cells Conclusions: Taken together, these data suggest that Mlkl but not Ripk3 deficiency impaired adipogenesis of 3T3-L1 cells by reducing the expression of pro-adipogenic factors and genes involved in fatty acid metabolism.
2022-10-10 | GSE201450 | GEO