RIPK1-dependent Apoptosis Research Articles

Influenza virus infection activates TAK1 to suppress RIPK3-independent apoptosis and RIPK1-dependent necroptosis

Many DNA viruses develop various strategies to inhibit cell death to facilitate their replication. However, whether influenza A virus (IAV), a fast-replicating RNA virus, attenuates cell death remains unknown. Here, we report that IAV infection induces TAK1 phosphorylation in a murine alveolar epithelial cell line (LET1) and a murine fibroblastoma cell line (L929). The TAK1-specific inhibitor 5Z-7-Oxzeneonal (5Z) and TAK1 knockout significantly enhance IAV-induced apoptosis, as evidenced by increased PARP, caspase-8, and caspase-3 cleavage. TAK1 inhibition also increases necroptosis as evidenced by increased RIPK1S166, RIPK3T231/S232, and MLKLS345 phosphorylation. Mechanistically, TAK1 activates IKK, which phosphorylates RIPK1S25 and inhibits its activation. TAK1 also activates p38 and its downstream kinase MK2, which phosphorylates RIPK1S321 but does not affect RIPK1 activation. Further investigation revealed that the RIPK1 inhibitor Nec-1 and RIPK1 knockout abrogate IAV-induced apoptosis and necroptosis; re-expression of wild-type but not kinase-dead (KD)-RIPK1 restores IAV-induced cell death. ZBP1 knockout abrogates IAV-induced cell death, whereas RIPK3 knockout inhibits IAV-induced necroptosis but not apoptosis. 5Z treatment enhances IAV-induced cell death and slightly reduces the inflammatory response in the lungs of H1N1 virus-infected mice and prolongs the survival of IAV-infected mice. Our study provides evidence that IAV activates TAK1 to suppress RIPK1-dependent apoptosis and necroptosis, and that RIPK3 is required for IAV-induced necroptosis but not apoptosis in epithelial cells.

The autophagy protein RUBCNL/PACER represses RIPK1 kinase-dependent apoptosis and necroptosis

ABSTRACT Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. Abbreviations: BAF: bafilomycin A1; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; siCtrl: small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a

The role of PPP1R3G in promoting necroptosis in ischemia reperfusion induced acute kidney injury

Recent discoveries have revealed necroptosis as a major contributor to the pathogenesis of acute kidney injury (AKI). Protein phosphatase 1 regulatory subunit 3G (PPP1R3G) has been linked to the essentiality of RIPK1-dependent apoptosis and type I necroptosis. Nonetheless, the involvement of PPP1R3G in the regulation of necroptosis during ischemia reperfusion-induced acute kidney injury (IR-AKI) has yet to be elucidated. Here, we investigated the role of PPP1R3G in the regulation of necroptosis in IR using an in vitro cellular I/R model and in vivo IRI-AKI mouse model. Primary proximal tubular cells (PTCs) from C57BL/6 and Ppp1r3g−/− mice were cultured and cell I/R was induced through hypoxia/reoxygenation. Cell viability and necroptosis were analyzed using CellTiter-Glo assay and Annexin V-Cy3/ SYTOX green staining, respectively. The expression levels of necroptosis, oxidative stress, and inflammation factors were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. In the in vivo IRI-AKI model, bilateral renal pedicles clamping was conducted in both C57BL/6 and Ppp1r3g−/− mice. The kidney injury and function were evaluated by plasma creatine, BUN, and KIM-1 levels and GFR values, respectively. Kidney morphological evidence of injury was assessed by PAS staining and cell death by immunofluorescent staining. We found that knockout of PPP1R3G in the primary tubular cells significantly preserved cell viability and attenuated tubular cell death. This protective effect was associated with the reduced expression and phosphorylation of necroptosis factors (RIP1, RIP3, and MLKL), and inflammatory factors (IL-6, IL-10 and TNF-α) in I/R injury cells. Deletion of PPP1R3G attenuated the I/R induced kidney injury indicated by 54% decrease in the plasma creatine (2.28±0.12mg/dL vs. 1.09±0.07mg/dL, p<0.0001, n=7) and 45% higher GFR values than WT mice at day 3 after reperfusion. Histology showed less diffuse renal tubular necrosis and casts comprising necrotic cells and debris in Ppp1r3g−/− mice, as well as 60% less cell death compared to WT mice. Moreover, the application of chemical compounds to prevent inhibitory phosphorylation of RIPK1 significantly reinstated cell death in Ppp1r3g−/− cells. In conclusion, our findings demonstrate, for the first time, that Ppp1r3g-mediated necroptosis significantly promotes IRI-AKI. Therefore, targeting Ppp1r3g to reduce necroptosis in the kidney holds promising potential for significant clinical benefits. National Institutes of Health Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers.

Poor survival and lack of treatment response in glioblastoma (GBM) is attributed to the persistence of glioma stem cells (GSCs). To identify novel therapeutic approaches, we performed CRISPR/Cas9 knockout screens and discovered TGFβ activated kinase (TAK1) as a selective survival factor in a significant fraction of GSCs. Loss of TAK1 kinase activity results in RIPK1-dependent apoptosis via Caspase-8/FADD complex activation, dependent on autocrine TNFα ligand production and constitutive TNFR signaling. We identify a transcriptional signature associated with immune activation and the mesenchymal GBM subtype to be a characteristic of cancer cells sensitive to TAK1 perturbation and employ this signature to accurately predict sensitivity to the TAK1 kinase inhibitor HS-276. In addition, exposure to pro-inflammatory cytokines IFNγ and TNFα can sensitize resistant GSCs to TAK1 inhibition. Our findings reveal dependency on TAK1 kinase activity as a novel vulnerability in immune-activated cancers, including mesenchymal GBMs that can be exploited therapeutically.

Retraction: Reduced levels of A20 protein prompted RIPK1-dependent apoptosis and blood-brain barrier breakdown during cerebral ischemia reperfusion injury.

Retraction: Reduced levels of A20 protein prompted RIPK1-dependent apoptosis and blood-brain barrier breakdown during cerebral ischemia reperfusion injury.

Small molecule activators of TAK1 promotes its activity-dependent ubiquitination and TRAIL-mediated tumor cell death

TAK1 is a key modulator of both NF-κB signaling and RIPK1. In TNF signaling pathway, activation of TAK1 directly mediates the phosphorylation of IKK complex and RIPK1. In a search for small molecule activators of RIPK1-mediated necroptosis, we found R406/R788, two small molecule analogs that could promote sustained activation of TAK1. Treatment with R406 sensitized cells to TNF-mediated necroptosis and RIPK1-dependent apoptosis by promoting sustained RIPK1 activation. Using click chemistry and multiple biochemical binding assays, we showed that treatment with R406 promotes the activation of TAK1 by directly binding to TAK1, independent of its original target Syk kinase. Treatment with R406 promoted the ubiquitination of TAK1 and the interaction of activated TAK1 with ubiquitinated RIPK1. Finally, we showed that R406/R788 could promote the cancer-killing activities of TRAIL in vitro and in mouse models. Our studies demonstrate the possibility of developing small molecule TAK1 activators to potentiate the effect of TRAIL as anticancer therapies.

Reduced levels of A20 protein prompted RIPK1-dependent apoptosis and blood-brain barrier breakdown during cerebral ischemia reperfusion injury.

Blood-brain barrier (BBB) leakage is an important cause of the exacerbation of pathological features of cerebral ischemia reperfusion injury (CIRI). However, the specific mechanism of BBB leakage is not clear. It was found that the CIRI resulted in RIPK1 activation and subsequent RIPK1-dependent apoptosis (RDA). Inhibition of RIPK1 significantly reduced BBB breakdown and brain damage. The aim of this study is to investigate the mechanism of RIPK1 in the BBB leakage during CIRI. It was discovered by proteomics that autophagy activation resulting from ischemia and reperfusion significantly downregulated the level of A20 protein. A20 is an important protein that regulates RIPK1 and RDA. It was hypothesized that activation of autophagy caused by ischemic reperfusion led to a decrease in A20 protein, which, in turn, caused the activation of RIPK1 and the occurrence of RDA, leading to leakage of the BBB. The findings in this study revealed the role of RIPK1 in the cell death and BBB leakage upon cerebral ischemia reperfusion injury, and these findings provide a novel perspective for the treatment of ischemic reperfusion.

IKK promotes naïve T cell survival by repressing RIPK1-dependent apoptosis and activating NF-κB.

The inhibitor of κB kinase (IKK) complex regulates the activation of the nuclear factor κB (NF-κB) family of transcription factors. In addition, IKK represses extrinsic cell death pathways dependent on receptor-interacting serine/threonine-protein kinase 1 (RIPK1) by directly phosphorylating this kinase. Here, we showed that peripheral naïve T cells in mice required the continued expression of IKK1 and IKK2 for their survival; however, the loss of these cells was only partially prevented when extrinsic cell death pathways were blocked by either deleting Casp8 (which encodes the apoptosis-inducing caspase 8) or inhibiting the kinase activity of RIPK1. Inducible deletion of Rela (which encodes the NF-κB p65 subunit) in mature CD4+ T cells also resulted in loss of naïve CD4+ T cells and in reduced abundance of the interleukin-7 receptor (IL-7R) encoded by the NF-κB target Il7r, revealing an additional reliance upon NF-κB for the long-term survival of mature T cells. Together, these data indicate that the IKK-dependent survival of naïve CD4+ T cells depends on both repression of extrinsic cell death pathways and activation of an NF-κB-dependent survival program.

From PERK to RIPK1: Design, synthesis and evaluation of novel potent and selective necroptosis inhibitors.

Receptor-Interacting serine/threonine-Protein Kinase 1 (RIPK1) emerged as an important driver of inflammation and, consequently, inflammatory pathologies. The enzymatic activity of RIPK1 is known to indirectly promote inflammation by triggering cell death, in the form of apoptosis, necroptosis and pyroptosis. Small molecule Receptor-Interacting serine/threonine-Protein Kinase 1 inhibitors have therefore recently entered clinical trials for the treatment of a subset of inflammatory pathologies. We previously identified GSK2656157 (GSK'157), a supposedly specific inhibitor of protein kinase R (PKR)-like ER kinase (PERK), as a much more potent type II Receptor-Interacting serine/threonine-Protein Kinase 1 inhibitor. We now performed further structural optimisation on the GSK'157 scaffold in order to develop a novel class of more selective Receptor-Interacting serine/threonine-Protein Kinase 1 inhibitors. Based on a structure-activity relationship (SAR) reported in the literature, we anticipated that introducing a substituent on the para-position of the pyridinyl ring would decrease the interaction with PERK. Herein, we report a series of novel GSK'157 analogues with different para-substituents with increased selectivity for Receptor-Interacting serine/threonine-Protein Kinase 1. The optimisation led to UAMC-3861 as the best compound of this series in terms of activity and selectivity for Receptor-Interacting serine/threonine-Protein Kinase 1 over PERK. The most selective compounds were screened in vitro for their ability to inhibit RIPK1-dependent apoptosis and necroptosis. With this work, we successfully synthesised a novel series of potent and selective type II Receptor-Interacting serine/threonine-Protein Kinase 1 inhibitors based on the GSK'157 scaffold.

Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells.

Sapoviruses belonging to the genus Sapovirus within the family Caliciviridae are commonly responsible for severe acute gastroenteritis in both humans and animals. Caliciviruses are known to induce intrinsic apoptosis in vitro and in vivo, however, calicivirus-induced necroptosis remains to be fully elucidated. Here, we demonstrate that infection of porcine kidney LLC-PK cells with porcine sapovirus (PSaV) Cowden strain as a representative of caliciviruses induces receptor-interacting protein kinase 1 (RIPK1)-dependent necroptosis and acts as proviral compared to the antiviral function of PSaV-induced apoptosis. Infection of LLC-PK cells with PSaV Cowden strain showed that the interaction of phosphorylated RIPK1 (pRIPK1) with RIPK3 (pRIPK3), mixed lineage kinase domain-like protein (pMLKL) increased in a time-dependent manner, indicating induction of PSaV-induced RIPK1-dependent necroptosis. Interfering of PSaV-infected cells with each necroptotic molecule (RIPK1, RIPK3, or MLKL) by treatment with each specific chemical inhibitor or knockdown with each specific siRNA significantly reduced replication of PSaV but increased apoptosis and cell viability, implying proviral action of PSaV-induced necroptosis. In contrast, treatment of PSaV-infected cells with pan-caspase inhibitor Z-VAD-FMK increased PSaV replication and necroptosis, indicating an antiviral action of PSaV-induced apoptosis. These results suggest that PSaV-induced RIPK1-dependent necroptosis and apoptosis‒which have proviral and antiviral effects, respectively‒counterbalanced each other in virus-infected cells. Our study contributes to understanding the nature of PSaV-induced necroptosis and apoptosis and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections.

Necrostatin-1 Alleviates Lung Ischemia-Reperfusion Injury via Inhibiting Necroptosis and Apoptosis of Lung Epithelial Cells.

Lung ischemia-reperfusion injury (LIRI) is associated with many diseases, including primary graft dysfunction after lung transplantation, and has no specific and effective therapies. Necroptosis contributes to the pathogenesis of ischemia-reperfusion injury. Necrostatin-1 (Nec-1), the necroptosis inhibitor targeting RIPK1, has been reported to alleviate ischemia-reperfusion injury in various organs. However, the underlying mechanism of Nec-1 in LIRI remains unclear. In this paper, an in vivo LIRI model was built up by left lung hilar clamping in mice, and an in vitro cold ischemia-reperfusion (CI/R) model using BEAS-2B cells was applied to mimic the lung transplantation setting. We found Nec-1 significantly alleviated ischemia-reperfusion-induced lung injury, cytokine releasing, and necroptosis of epithelial cells in mouse lungs. In vitro, Nec-1 also mitigated CI/R-induced cell death and inflammatory responses in BEAS-2B cells, and these protective effects were achieved by simultaneously inhibiting the formation of necrosome and RIPK1-dependent apoptosis. However, Nec-1 decreased the necrosome number but increased the apoptosis level in lung tissues after ischemia reperfusion. We further clarified that Nec-1 could also attenuate lung injury by promoting neutrophil apoptosis from flow cytometry. In conclusion, Nec-1 alleviated lung ischemia-reperfusion injury by inhibiting necroptosis and apoptosis of epithelial cells and promoting the apoptosis of neutrophils. Thus, Nec-1 could be a promising medication against primary graft dysfunction after lung transplantation.

Inhibition of TWEAK/Tnfrsf12a axis protects against acute liver failure by suppressing RIPK1-dependent apoptosis

Acute liver failure (ALF) is a severe clinical syndrome characterized by massive death of hepatocytes in a short time, resulting in coagulopathy and hepatic encephalopathy, with a high mortality in patients without pre-existing liver disease. Effective treatment of ALF is currently limited to liver transplantation, highlighting the need for new target therapies. Here, we found that expression of hepatic tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor tumor necrosis factor receptor superfamily member 12A (Tnfrsf12a) were significantly increased during ALF induced by thioacetamide (TAA) or acetaminophen (APAP). Inhibition of TWEAK/Tnfrsf12a axis markedly attenuated TAA or APAP-induced ALF. Moreover, our results demonstrated that TWEAK/Tnfrsf12a axis induced receptor-interacting protein kinase 1 (RIPK1)-dependent apoptosis of hepatocytes, instead of necroptosis or pyroptosis. Notably, hepatic TNFRSF12A and TWEAK levels were also significantly increased in liver biopsies from ALF patients. In summary, our results demonstrate that during ALF, TWEAK/Tnfrsf12a axis activates RIPK1 in hepatocytes, leading to RIPK1-dependent apoptosis and subsequent liver injury. Therefore, inhibition of either TWEAK/Tnfrsf12a axis or RIPK1-dependent apoptosis attenuates liver injury, providing a new potential therapeutic target for the treatment of ALF.

MascRNA promotes macrophage apoptosis, inhibits osteoclast differentiation and attenuates disease progression in a murine model of arthritis

Macrophages play a crucial role in the pathogenesis of rheumatoid arthritis (RA) and have been considered as a therapeutic target of this disease. Here we show that mascRNA, a tRNA-like cytoplasmic small noncoding RNA, promoted RIPK1-dependent apoptosis (RDA) in RAW267.4 macrophages in response to the TAK1 inhibitor 5Z-7-oxozeaenol (5Z-7) alone as well as in combination with TNF. Moreover, mascRNA suppressed RANKL-induced expression of osteoclast marker genes and attenuated RANKL signaling. Using a murine model of collagen-induced arthritis (CIA), we demonstrated that mascRNA, administered either alone or in combination with 5Z-7, alleviated joint inflammation in CIA mice. Thus, mascRNA might be a promising agent for the treatment of RA.

TAB2 deficiency induces dilated cardiomyopathy by promoting RIPK1-dependent apoptosis and necroptosis.

Mutations in TGF-β–activated kinase 1 binding protein 2 (TAB2) have been implicated in the pathogenesis of dilated cardiomyopathy and/or congenital heart disease in humans, but the underlying mechanisms are currently unknown. Here, we identified an indispensable role for TAB2 in regulating myocardial homeostasis and remodeling by suppressing receptor-interacting protein kinase 1 (RIPK1) activation and RIPK1-dependent apoptosis and necroptosis. Cardiomyocyte-specific deletion of Tab2 in mice triggered dilated cardiomyopathy with massive apoptotic and necroptotic cell death. Moreover, Tab2-deficient mice were also predisposed to myocardial injury and adverse remodeling after pathological stress. In cardiomyocytes, deletion of TAB2 but not its close homolog TAB3 promoted TNF-α–induced apoptosis and necroptosis, which was rescued by forced activation of TAK1 or inhibition of RIPK1 kinase activity. Mechanistically, TAB2 critically mediates RIPK1 phosphorylation at Ser321 via a TAK1-dependent mechanism, which prevents RIPK1 kinase activation and the formation of RIPK1-FADD-caspase-8 apoptotic complex or RIPK1-RIPK3 necroptotic complex. Strikingly, genetic inactivation of RIPK1 with Ripk1-K45A knockin effectively rescued cardiac remodeling and dysfunction in Tab2-deficient mice. Together, these data demonstrated that TAB2 is a key regulator of myocardial homeostasis and remodeling by suppressing RIPK1-dependent apoptosis and necroptosis. Our results also suggest that targeting RIPK1-mediated cell death signaling may represent a promising therapeutic strategy for TAB2 deficiency–induced dilated cardiomyopathy.

The potent inhibitory role of suppressing TBK1 in RIPK1 associated cerebral ischemia-reperfusion injury

The pathological mechanism of cell death features in cerebral ischemia-reperfusion injury (CIRI) was complicated. The occurrence of various cell death pathways during the progression of ischemia/reperfusion injury promoted complex further neuroinflammation. RIPK1, receptor interacting protein kinase 1, was convinced to be involved in both necroptosis and apoptosis, which is a special RIPK1-dependent apoptosis. More evidences indicated the physiological role of RIPK1 in necroptosis, apoptosis and also autophagy.In this study, we elucidated the RIPK1 exhibited characterization in various cell death pathways in time-course dependent feature. The necroptosis occupied dominant neuron death within 24 h after ischemia/reperfusion injury. However, the neuronal death feature seemed turned to apoptosis 24 h after reperfusion.In this study, it was also found that TBK1 (TANK binding kinase 1) played as suppressor in the regulation of kinase activity of RIPK1. This result might provide a potential approach in mediating the kinase activity of RIPK1 in clinic.

RIPK1 dephosphorylation and kinase activation by PPP1R3G/PP1\u03b3 promote apoptosis and necroptosis

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of inflammation and cell death. Many sites on RIPK1, including serine 25, are phosphorylated to inhibit its kinase activity and cell death. How these inhibitory phosphorylation sites are dephosphorylated is poorly understood. Using a sensitized CRISPR whole-genome knockout screen, we discover that protein phosphatase 1 regulatory subunit 3G (PPP1R3G) is required for RIPK1-dependent apoptosis and type I necroptosis. Mechanistically, PPP1R3G recruits its catalytic subunit protein phosphatase 1 gamma (PP1γ) to complex I to remove inhibitory phosphorylations of RIPK1. A PPP1R3G mutant which does not bind PP1γ fails to rescue RIPK1 activation and cell death. Furthermore, chemical prevention of RIPK1 inhibitory phosphorylations or mutation of serine 25 of RIPK1 to alanine largely restores cell death in PPP1R3G-knockout cells. Finally, Ppp1r3g−/− mice are protected from tumor necrosis factor-induced systemic inflammatory response syndrome, confirming the important role of PPP1R3G in regulating apoptosis and necroptosis in vivo.

Bclaf1 regulates c-FLIP expression and protects cells from TNF-induced apoptosis and tissue injury.

TNF stimulation generates pro-survival signals through activation of NF-κB that restrict the build-in death signaling triggered by TNF. The competition between TNF-induced survival and death signals ultimately determines the fate of a cell. Here, we report the identification of Bclaf1 as a novel component of the anti-apoptotic program of TNF. Bclaf1 depletion in multiple cells sensitizes cells to TNF-induced apoptosis but not to necroptosis. Bclaf1 exerts its anti-apoptotic function by promoting the transcription of CFLAR, a caspase 8 antagonist, downstream of NF-κB activation. Bclaf1 binds to the p50 subunit of NF-κB, which is required for Bclaf1 to stimulate CFLAR transcription. Finally, in Bclaf1 siRNA administered mice, TNF-induced small intestine injury is much more severe than in control mice with aggravated signs of apoptosis and pyroptosis. These results suggest Bclaf1 is a key regulator in TNF-induced apoptosis, both invitro and invivo.

NEK1-mediated retromer trafficking promotes blood\u2013brain barrier integrity by regulating glucose metabolism and RIPK1 activation

Loss-of-function mutations in NEK1 gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS. Here we show that NEK1 regulates retromer-mediated endosomal trafficking by phosphorylating VPS26B. NEK1 deficiency disrupts endosomal trafficking of plasma membrane proteins and cerebral proteome homeostasis to promote mitochondrial and lysosomal dysfunction and aggregation of α-synuclein. The metabolic and proteomic defects of NEK1 deficiency disrupts the integrity of blood–brain barrier (BBB) by promoting lysosomal degradation of A20, a key modulator of RIPK1, thus sensitizing cerebrovascular endothelial cells to RIPK1-dependent apoptosis and necroptosis. Genetic inactivation of RIPK1 or metabolic rescue with ketogenic diet can prevent postnatal lethality and BBB damage in NEK1 deficient mice. Inhibition of RIPK1 reduces neuroinflammation and aggregation of α-synuclein in the brains of NEK1 deficient mice. Our study identifies a molecular mechanism by which retromer trafficking and metabolism regulates cerebrovascular integrity, cerebral proteome homeostasis and RIPK1-mediated neuroinflammation.

ABIN-1 is a key regulator in RIPK1-dependent apoptosis (RDA) and necroptosis, and ABIN-1 deficiency potentiates necroptosis-based cancer therapy in colorectal cancer

ABIN-1, also called TNIP1, is an ubiquitin-binding protein that serves an important role in suppressing RIPK1-independent apoptosis, necroptosis, and NF-κB activation. However, the involvement of ABIN-1 in the regulation of RIPK1-dependent apoptosis (RDA) is unknown. In this study, we found that poly(I:C) + TAK1 inhibitor 5Z-7-oxozeaenol (P5) concurrently induces RDA and necroptosis in Abin-1−/−, but not in Abin-1+/+ mouse embryonic fibroblasts (MEFs). Upon P5 stimulation, cells initially die by necroptosis and subsequently by RDA. Furthermore, we explored the therapeutic effect of ABIN-1 deficiency in necroptosis-based cancer therapy in colorectal cancer (CRC). We found that poly(I:C) + 5Z-7-oxozeaenol + IDN-6556 (P5I) yields a robust pro-necroptosis response, and ABIN-1 deficiency additionally enhances this P5I-induced necroptosis. Moreover, phase I/II cIAP inhibitor birinapant with clinical caspase inhibitor IDN-6556 (BI) alone and 5-fluorouracil with IDN-6556 (FI) alone are sufficient to induce necroptotic cell death in CRC cells by promoting auto-secretion of tumor necrosis factor (TNF); ABIN-1 deficiency amplifies the BI- or FI-induced necroptosis. Two independent xenograft experiments using HT-29 or COLO205 cells show that both BI and P5I remarkably inhibit tumor growth via necroptosis activation. For poly(I:C)-induced cell death, the sensitizing effect of ABIN-1 deficiency on cell death may be attributed to increased expression of TLR3. In TNF-induced necroptosis, ABIN-1 deficiency increases TNF-induced RIPK1 polyubiquitination by reducing the recruitment of ubiquitin-editing enzyme A20 to the TNFR1 signaling complex and induces more TNF secretion in CRC cells upon pro-necroptosis stimulation. With this combined data, ABIN-1 deficiency promotes greater sensitization of CRC cells to necroptosis.

Thymoquinone reduced RIPK1-dependent apoptosis caused by valproic acid in rat brain

Thymoquinone reduced RIPK1-dependent apoptosis caused by valproic acid in rat brain