Transcriptome analysis of TNFα or necroptosis-induced stimulation in the NCI-H441 cell line with RIPK3 knocked down by siRNA.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE270211 | GEO | 2026/04/01
REPOSITORIES: GEO
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Project description:Transcriptome analysis of TNFα or necroptosis-induced stimulation in the NCI-H441 cell line with RIPK3 knocked down by siRNA.
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Project description:Extracellular vesicles (EVs) are important mechanisms used by cells to release biomolecules. A common necroptosis effector— mixed lineage kinase like (MLKL)--- was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. We performed mass spectrometry-based proteomics on EVs released by healthy or necroptotic cells. Necroptosis increased the number of EVs released and altered the protein contents within the EVs. Comparing to EVs released by healthy cells, EVs released during necroptosis contained markedly higher number of unique proteins. Receptor interacting protein kinase 3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, MEFs derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis-associated EVs was sensitive to Ca2+ depletion or lysosomal disruption. Neither treatments affected the RIPK3-mediated MLKL phosphorylation. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.
2022-08-14 | PXD031224 | Pride
Project description:Necroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damage and impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic and persistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated because proteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining the role of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributes to the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing that necroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docile strain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads are not altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, we identified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. This was not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function of RIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to an impaired antiviral immune response and abrogated clearance of chronic LCMV infection.
2023-03-12 | GSE224066 | GEO
Project description:Pathogen recognition receptors and TNF superfamily members engage Receptor Interacting Serine/threonine Kinase-3 (RIPK3) to activate programmed cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 signalling is not fully understood. Using mass-spectrometry, we identified a novel ubiquitylation site on murine RIPK3 beyond the RIP homotypic interaction motif (RHIM) on K469. Complementation of RIPK3-deficient cells with a Ripk3K469R mutant demonstrated that the decoration of RIPK3 K469 by ubiquitin limits both RIPK3-mediated caspase-8 activation and apoptotic killing, in addition to RIPK3 autophosphorylation and MLKL-mediated necroptosis. Unexpectedly, the overall ubiquitylation of mutant RIPK3K469R was enhanced, which largely resulted from additional RIPK3 ubiquitylation on K359. Loss of RIPK3 K359 ubiquitylation reduced RIPK3K469R hyper-ubiquitylation and limited the ability of Ripk3K469R/K469R to trigger enhanced killing. Ripk3K469R/K469R mice challenged with Salmonella displayed increased bacterial loads in the spleen and liver, with reduced IFN serum levels. Therefore, RIPK3 K469 ubiquitylation can function to prevent RIPK3 ubiquitylation on alternate lysine residues, which otherwise promote RIPK3 oligomerization and consequent cell death signalling.
2022-08-12 | PXD026533 | Pride
Project description:Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL. While the precise molecular cue that prompts RIPK3 to activate MLKL is incompletely understood, it is known that RIPK3 is highly regulated by phosphorylation. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, extensive mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than the wild-type RIPK3, mutation of T224, S227, and RHIM in RIPK3 attenuated or compromised their abilities to mediate necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL.
2022-07-30 | PXD033488 | Pride
Project description:Epithelial Gab1 calibrates RIPK3-dependent necroptosis to prevent intestinal inflammation
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Project description:Necroptosis is a programmed lytic cell death involving active cytokine production and plasma membrane rupture through distinct signaling cascades. However, it remains challenging to delineate this inflammatory cell death pathway at specific signaling nodes with spatiotemporal accuracy. To address this challenge, we developed an optogenetic system, termed Light-activatable Receptor-Interacting Protein Kinase 3 or La-RIPK3, to enable ligand-free, optical induction of RIPK3 oligomerization. La-RIPK3 activation dissects RIPK3-centric lytic cell death through the induction of RIPK3-containing necrosome, which mediates cytokine production and plasma membrane rupture. Bulk RNA-Seq analysis reveals that RIPK3 oligomerization results in partially overlapped gene expression compared to pharmacological induction of necroptosis. However, La-RIPK3 activates a group of genes likely regulated by RIPK3 kinase-independent processes. Using patterned light stimulation delivered by a spatial light modulator, we demonstrate precise spatiotemporal control of necroptosis in La-RIPK3-transduced HT-29 cells. Optogenetic control of proinflammatory lytic cell death could lead to the development of innovative experimental strategies to finetune the immune landscape for disease intervention.
2024-06-05 | GSE268650 | GEO
Project description:RIPK3 is a key regulator of necroptosis and inflammation, yet the distinct contributions of its kinase-dependent and -independent functions remain poorly understood. Here, we report the generation and characterization of RIPK3 D143N mutant mice, a novel model that selectively ablates kinase activity without inducing apoptosis or altering protein expression. Unlike previously described RIPK3 mutants (D161N/K51A), the D143N variant fully rescues Casp8-deficient embryonic lethality while effectively blocking necroptosis induced by multiple stimuli. In TNF-a-induced SIRS, we demonstrated that RIPK3's non-kinase activity exacerbates inflammation and tissue damage through JAK-STAT activation. Similarly, the kinase-independent RIPK3 activity promoted influenza-induced pathogenesis. Our findings establish the D143N mutant as a unique tool for dissecting non-catalytic functions of RIPK3, and provide critical insights for targeting RIPK3 for disease treatment.
2026-03-11 | GSE305184 | GEO
Project description:Pathological TAR DNA-binding protein-43 (TDP-43) is a defining feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer’s disease (AD). However, the mechanism by which TDP-43 pathology disrupts microglial function and drives neuroinflammation remain unclear. In this study, we demonstrated that cytoplasmic mis-localized TDP-43 exacerbated neuroinflammation, induced cell death, and impaired phagocytic function in microglial cells, primarily through receptor interacting serine/threonine kinase 3 (RIPK3)-dependent necroptosis. Pharmacological inhibition of RIPK3 with GSK872 markedly attenuated these pathological effects in vitro. These findings were further corroborated in a murine model with cytoplasmic TDP-43 mis-localization, where GSK872 treatment remarkably alleviated neuroinflammation and restored cognitive deficits. Mechanistically, our findings indicate that the nuclear depletion of TDP-43, resulted from its cytoplasmic mis-localization, impairs its ability to transcriptionally repress the Ripk3 gene, subsequently leading to RIPK3 upregulation and activation of RIPK3-dependent necroptosis. Collectively, our findings establish RIPK3-dependent necroptosis as a critical driver of TDP-43 pathology-mediated neuroinflammation and identified necroptosis as a promising therapeutic target in TDP-43-associated neurodegenerative disorders.
2026-01-04 | GSE315250 | GEO
Project description:Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a well-known inducer of apoptosis via formation of the primary death-inducing signaling complex (TRAIL-DISC) at the level of membrane death receptors (DR4 and DR5) which recruit successively FADD and caspase-8. TRAIL can also induce necroptosis when caspases are inhibited. Necroptosis is a regulated cell death dependent on the formation of a cytosolic necrosome complex which includes RIPK1, RIPK3 and MLKL proteins. Elucidating the molecular mechanisms involved in TRAIL-induced necroptosis might provide new insights into the TRAIL death signaling pathway. Here, we report the analysis by mass spectrometry of endogenous RIPK3-dependent necrosome complex constituents upon necroptosis induced by TRAIL/z-VAD/Birinapant (TzB) in HT29 cells. Besides characterization of RIPK1, RIPK3, MLKL, FADD, caspase-8, we find TRIM21 as a new constituent of the necrosome complex. Moreover RIPK1, RIPK3, MLKL, P-MLKL, FADD, caspase-8 and TRIM21 are also found associated to the native TRAIL-DISC upon TzB stimulation showing initiation of the necrotic pathway at the level of TRAIL death receptors in HT29 cells. Finally, TRIM21 may positively modulate necroptosis induction by downregulating NF-kB activation.
2021-03-18 | PXD003383 | Pride