RIP3 and MLKL IP-SWATH mass spectrometry
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Chuanqi Zhong
PROVIDER: PXD067729 | iProX | Tue Aug 26 00:00:00 BST 2025
REPOSITORIES: iProX
Similar Datasets
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: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:OBJECTIVES: To detect the expression levels of MLKL and p-MLKL, and explore its potential roles in inflammatory cell infiltration, angiogenesis, and bone destruction, in human and mouse periapical lesions. METHODS: Forty-six human periapical tissues, including periapical granulomas (PGs, n =26), radicular cysts (RCs, n =20), and eight healthy control tissues were collected. Samples were fixed and analyzed by HE staining, RNA-Seq, western blot, and immunohistochemical staining. A periapical lesion mouse model was induced by pulp exposure in the first lower molars of 15 C57BL/6J mice. After lesion induction, the mice were sacrificed on days 0, 21, and 35. Mandibles were harvested for microcomputed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. Double immunofluorescence was utilized to assess the colocalization of phosphorylated MLKL (p-MLKL) with CD34, matrix metalloproteinase-9 (MMP-9), and Cathepsin K (CTSK) in human and mouse periapical lesions. RESULTS: RNA-Seq analysis showed that, in comparison with healthy gingiva tissues, MLKL was more significantly upregulated in periapical lesions (P-value < 0.05). Immunohistochemistry staining showed that, MLKL and p-MLKL were significantly overexpressed in the RC and PG groups compared with the control group (P-value < 0.05). However, the difference between the RC and PG groups was insignificant (P-value > 0.05). p-MLKL-positive cells were mainly lymphocytes, epithelial cells, and endothelial cells around the vascular wall. In mouse periapical lesions, the expression levels of p-MLKL were positively correlated with the bone defect area and tartrate-resistant acid phosphatase-positive (TRAP+) cell amounts (R2=0.4108, P-value < 0.05; R2=0.5668, P-value < 0.05, respectively). The double-labeling analysis showed that p-MLKL colocalized with CD34 and MMP-9 in human samples, and with CTSK adjacent to the bone in mouse periapical lesions. CONCLUSION: MLKL and p-MLKL were overexpressed in human periapical lesions. p-MLKL exhibited a close relationship with angiogenesis and alveolar bone resorption.
2024-10-24 | GSE246108 | GEO
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
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: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:Individuals with genetic elimination of MLKL exhibit an increased susceptibility to neurodegenerative diseases like Alzheimer’s disease (AD). However, the specific mechanism behind this relationship is not yet fully understood. Here, we initially observed significant compromise in autophagy in the brains of MLKL knockout (KO) mice, as evidenced by the down-regulation of autophagy-initiation proteins Beclin1 and ULK1. To elucidate the mechanism by which MLKL, primarily a cytosolic protein, regulates the protein levels of Beclin1 and ULK1, we performed a proteomic screening of MLKL-association proteins and identified UBA52 as the binding partner under physiological conditions. Loss of MLKL induces a decrease in ubiquitin levels by preventing UBA52 from producing ubiquitin and the C-terminal ribosomal protein L40. Furthermore, we demonstrated that in the brain, the deubiquitinase (DUB) USP7 mediates the processing of UBA52, which is regulated by MLKL. Knock down (KD) of USP7 or UBA52 showed similar effects on autophagy and ubiquitin levels as MLKL KD/KO. Moreover, our results indicated that the reduction of Beclin1 and ULK1 upon MLKL loss is attributed to a decrease in their lysine 63 (K63)-linked polyubiquitination. Additionally, single-nucleus RNA sequencing revealed that the loss of MLKL resulted in the disruption of multiple neurodegenerative disease-related pathways, including those associated with AD. These results were consistent with the observation of cognitive impairment in MLKL KO mice and exacerbation of AD pathologies in an AD mouse model with MLKL deletion. Taken together, our findings demonstrate that MLKL-USP7-UBA52 signaling is required for autophagy in brain through maintaining ubiquitin homeostasis, and highlight the contribution of MLKL loss-induced ubiquitin deficits to the development of neurodegeneration. Thus, the maintenance of adequate levels of ubiquitin may provide a novel perspective to protect individuals from multiple neurodegenerative diseases through regulating autophagy.
2025-01-29 | GSE270139 | 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:Hematopoietic stem cells (HSCs) survive many types of cellular stress but often lose their regenerative and lymphopoietic capacities as a result. Such functional decline also occurs with age, and dysfunctional HSCs with impaired mitochondria accumulate during aging. However, the molecular link between HSC stress response and age-related functional decline remains poorly understood. Here we show that multiple stress responses converge on the RIPK3-MLKL axis to induce age-related changes in HSCs. The necroptosis effector MLKL is readily activated by inflammation and replication stress and accumulates in HSC mitochondria. Consequently, activated MLKL does not cause cell death but impairs HSC self-renewal and lymphoid differentiation. Such MLKL-mediated functional decline also occurs in HSCs during organismal aging, with activated MLKL primarily mediating age-related mitochondrial damage and reduced glycolytic flux. Collectively, our results establish the RIPK3-MLKL axis as a key mediator of HSC aging and identify a necroptosis-independent role of MLKL in mitochondrial damage.
2026-02-10 | GSE285110 | GEO
Project description:Hematopoietic stem cells (HSCs) survive many types of cellular stress but often lose their regenerative and lymphopoietic capacities as a result. Such functional decline also occurs with age, and dysfunctional HSCs with impaired mitochondria accumulate during aging. However, the molecular link between HSC stress response and age-related functional decline remains poorly understood. Here we show that multiple stress responses converge on the RIPK3-MLKL axis to induce age-related changes in HSCs. The necroptosis effector MLKL is readily activated by inflammation and replication stress and accumulates in HSC mitochondria. Consequently, activated MLKL does not cause cell death but impairs HSC self-renewal and lymphoid differentiation. Such MLKL-mediated functional decline also occurs in HSCs during organismal aging, with activated MLKL primarily mediating age-related mitochondrial damage and reduced glycolytic flux. Collectively, our results establish the RIPK3-MLKL axis as a key mediator of HSC aging and identify a necroptosis-independent role of MLKL in mitochondrial damage.
2026-02-10 | GSE285109 | GEO