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1.
Proc Natl Acad Sci U S A ; 117(9): 4959-4970, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071228

RESUMO

Apoptosis and necroptosis are two regulated cell death mechanisms; however, the interaction between these cell death pathways in vivo is unclear. Here we used cerebral ischemia/reperfusion as a model to investigate the interaction between apoptosis and necroptosis. We show that the activation of RIPK1 sequentially promotes necroptosis followed by apoptosis in a temporally specific manner. Cerebral ischemia/reperfusion insult rapidly activates necroptosis to promote cerebral hemorrhage and neuroinflammation. Ripk3 deficiency reduces cerebral hemorrhage and delays the onset of neural damage mediated by inflammation. Reduced cerebral perfusion resulting from arterial occlusion promotes the degradation of TAK1, a suppressor of RIPK1, and the transition from necroptosis to apoptosis. Conditional knockout of TAK1 in microglial/infiltrated macrophages and neuronal lineages sensitizes to ischemic infarction by promoting apoptosis. Taken together, our results demonstrate the critical role of necroptosis in mediating neurovascular damage and hypoperfusion-induced TAK1 loss, which subsequently promotes apoptosis and cerebral pathology in stroke and neurodegeneration.

2.
Nature ; 577(7788): 109-114, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31827280

RESUMO

Activation of RIPK1 controls TNF-mediated apoptosis, necroptosis and inflammatory pathways1. Cleavage of human and mouse RIPK1 after residues D324 and D325, respectively, by caspase-8 separates the RIPK1 kinase domain from the intermediate and death domains. The D325A mutation in mouse RIPK1 leads to embryonic lethality during mouse development2,3. However, the functional importance of blocking caspase-8-mediated cleavage of RIPK1 on RIPK1 activation in humans is unknown. Here we identify two families with variants in RIPK1 (D324V and D324H) that lead to distinct symptoms of recurrent fevers and lymphadenopathy in an autosomal-dominant manner. Impaired cleavage of RIPK1 D324 variants by caspase-8 sensitized patients' peripheral blood mononuclear cells to RIPK1 activation, apoptosis and necroptosis induced by TNF. The patients showed strong RIPK1-dependent activation of inflammatory signalling pathways and overproduction of inflammatory cytokines and chemokines compared with unaffected controls. Furthermore, we show that expression of the RIPK1 mutants D325V or D325H in mouse embryonic fibroblasts confers not only increased sensitivity to RIPK1 activation-mediated apoptosis and necroptosis, but also induction of pro-inflammatory cytokines such as IL-6 and TNF. By contrast, patient-derived fibroblasts showed reduced expression of RIPK1 and downregulated production of reactive oxygen species, resulting in resistance to necroptosis and ferroptosis. Together, these data suggest that human non-cleavable RIPK1 variants promote activation of RIPK1, and lead to an autoinflammatory disease characterized by hypersensitivity to apoptosis and necroptosis and increased inflammatory response in peripheral blood mononuclear cells, as well as a compensatory mechanism to protect against several pro-death stimuli in fibroblasts.

3.
Cell Death Dis ; 10(12): 923, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31801942

RESUMO

Upon necroptosis activation, receptor interacting serine/threonine kinase (RIPK)1 and RIPK3 form a necrosome complex with pseudokinase mixed lineage kinase-like (MLKL). Although protein phosphorylation is a key event for RIPK1 and RIPK3 activation in response to a necroptosis signal, relatively little is known about other factors that might regulate the activity of these kinases or necrosome formation. Through a gain-of-function screen with 546 kinases and 127 phosphatases, we identified casein kinase 1 gamma (CK1γ) as a candidate necroptosis-promoting factor. Here, we show that the decreased activity or amounts of CK1γ1 and CK1γ3, either by treatment with a chemical inhibitor or knockdown in cells, reduced TNFα-induced necroptosis. Conversely, ectopic expression of CK1γ1 or CK1γ3 exacerbated necroptosis, but not apoptosis. Similar to RIPK1 and RIPK3, CK1γ1 was also cleaved at Asp343 by caspase-8 during apoptosis. CK1γ1 and CK1γ3 formed a protein complex and were recruited to the necrosome harboring RIPK1, RIPK3 and MLKL. In particular, an autophosphorylated form of CK1γ3 at Ser344/345 was detected in the necrosome and was required to mediate the necroptosis. In addition, in vitro assays with purified proteins showed that CK1γ phosphorylated RIPK3, affecting its activity, and in vivo assays showed that the CK1γ-specific inhibitor Gi prevented abrupt death in mice with hypothermia in a model of TNFα-induced systemic inflammatory response syndrome. Collectively, these data suggest that CK1γ1 and CK1γ3 are required for TNFα-induced necroptosis likely by regulating RIPK3.

4.
Mol Cell ; 75(3): 457-468.e4, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31230815

RESUMO

Necroptosis, a cell death pathway mediated by the RIPK1-RIPK3-MLKL signaling cascade downstream of tumor necrosis factor α (TNF-α), has been implicated in many inflammatory diseases. Members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases are known for their anti-apoptotic, oncogenic, and anti-inflammatory roles. Here, we identify an unexpected role of TAM kinases as promoters of necroptosis, a pro-inflammatory necrotic cell death. Pharmacologic or genetic targeting of TAM kinases results in a potent inhibition of necroptotic death in various cellular models. We identify phosphorylation of MLKL Tyr376 as a direct point of input from TAM kinases into the necroptosis signaling. The oligomerization of MLKL, but not its membranal translocation or phosphorylation by RIPK3, is controlled by TAM kinases. Importantly, both knockout and inhibition of TAM kinases protect mice from systemic inflammatory response syndrome. In conclusion, this study discovers that immunosuppressant TAM kinases are promoters of pro-inflammatory necroptosis, shedding light on the biological complexity of the regulation of inflammation.


Assuntos
Proteínas Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Síndrome de Resposta Inflamatória Sistêmica/genética , c-Mer Tirosina Quinase/genética , Animais , Apoptose/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Síndrome de Resposta Inflamatória Sistêmica/patologia , Fator de Necrose Tumoral alfa/genética
5.
Proc Natl Acad Sci U S A ; 116(20): 9714-9722, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31048504

RESUMO

RIPK1 kinase has emerged as a promising therapeutic target for the treatment of a wide range of human neurodegenerative, autoimmune, and inflammatory diseases. This was supported by extensive studies which demonstrated that RIPK1 is a key mediator of apoptotic and necrotic cell death as well as inflammatory pathways. Furthermore, human genetic evidence has linked the dysregulation of RIPK1 to the pathogenesis of ALS as well as other inflammatory and neurodegenerative diseases. Importantly, unique allosteric small-molecule inhibitors of RIPK1 that offer high selectivity have been developed. These molecules can penetrate the blood-brain barrier, thus offering the possibility to target neuroinflammation and cell death which drive various neurologic conditions including Alzheimer's disease, ALS, and multiple sclerosis as well as acute neurological diseases such as stroke and traumatic brain injuries. We discuss the current understanding of RIPK1 regulatory mechanisms and emerging evidence for the pathological roles of RIPK1 in human diseases, especially in the context of the central nervous systems.

6.
Proc Natl Acad Sci U S A ; 116(8): 2996-3005, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718432

RESUMO

Necroptosis and ferroptosis are two distinct necrotic cell death modalities with no known common molecular mechanisms. Necroptosis is activated by ligands of death receptors such as tumor necrosis factor-α (TNF-α) under caspase-deficient conditions, whereas ferroptosis is mediated by the accumulation of lipid peroxides upon the depletion/or inhibition of glutathione peroxidase 4 (GPX4). The molecular mechanism that mediates the execution of ferroptosis remains unclear. In this study, we identified 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO), a compound known to inhibit heat shock protein 90 (HSP90), as an inhibitor of necroptosis that could also inhibit ferroptosis. We found that HSP90 defined a common regulatory nodal between necroptosis and ferroptosis. We showed that inhibition of HSP90 by CDDO blocked necroptosis by inhibiting the activation of RIPK1 kinase. Furthermore, we showed that the activation of ferroptosis by erastin increased the levels of lysosome-associated membrane protein 2a to promote chaperone-mediated autophagy (CMA), which, in turn, promoted the degradation of GPX4. Importantly, inhibition of CMA stabilized GPX4 and reduced ferroptosis. Our results suggest that activation of CMA is involved in the execution of ferroptosis.


Assuntos
Autofagia/genética , Glutationa Peroxidase/genética , Proteína 2 de Membrana Associada ao Lisossomo/genética , Chaperonas Moleculares/genética , Necrose/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Caspases/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Ferro/metabolismo , Ligantes , Peróxidos Lipídicos/genética , Peróxidos Lipídicos/metabolismo , Chaperonas Moleculares/metabolismo , Piperazinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Necrose Tumoral alfa/genética
7.
J Rehabil Med ; 51(1): 47-53, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30299526

RESUMO

OBJECTIVE: To evaluate the prevalence of, and risk factors for, cerebral palsy in Henan province, China. METHODS: The prevalence of cerebral palsy in children aged 0-6 years between September 2011 and September 2012 was investigated using a stratified-clustered-random sampling method. An age-, sex- , and residence-matched control group of typically developing children was recruited. Univariate analysis and multinomial logistic regression analysis were used to identify risk factors associated with cerebral palsy. RESULTS: The prevalence of cerebral palsy in Henan province was 2.37 per 1,000 live births. Risk factors included: moving into a newly painted room; complicating maternal diseases (infection, heart disease, hypertension, anaemia, diabetes, kidney disease) during pregnancy; high gravidity (> 3); foetal asphyxia; low birth-weight (< 2,500 g); and hypoxic-ischaemic encephalopathy. CONCLUSION: The prevalence of cerebral palsy in Henan province was 2.37 per 1,000 live births. Parents and clinicians should be aware of the risk factors for cerebral palsy.


Assuntos
Paralisia Cerebral/epidemiologia , Criança , Pré-Escolar , China/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Gravidez , Prevalência , Fatores de Risco
8.
Cell Death Differ ; 26(6): 1077-1088, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30341420

RESUMO

ABIN-1 (encoded by the gene Tnip1) is a ubiquitin-binding protein that can interact with ubiquitin-editing enzyme A20 (encoded by the gene TNFAIP3) to restrain the activation of necroptosis and NF-κB activation. Genetic variants in the genes Tnip1 and TNFAIP3 are both strongly associated with susceptibility to autoimmune chronic inflammatory diseases such as psoriasis vulgaris and systemic lupus erythematosus (SLE) in humans. Here we investigated the mechanism by which ABIN-1 regulated innate immune responses. We show that ABIN-1 heterozygosity sensitizes cells to antiviral response by mediating NF-κB-dependent and RIPK1-independent expression of pattern recognition molecules, including TLR3, RIG-I, and MDA5, in MEFs. Furthermore, we demonstrate that increased interaction of ABIN-1 and A20 with prolonged poly(I:C) stimulation of WT cells leads to A20-dependent reduction of ABIN-1 protein. Finally, we show that ABIN-1 heterozygosity sensitizes innate immune response of Abin-1+/- mice in vivo by promoting the production of proinflammatory cytokines, which can be blocked upon inhibition of RIPK1 kinase. Inhibition of RIPK1 kinase activity in vivo partially reduces the expression of MDA5, RIG-I, and caspase-11 in Abin-1+/- mice but not in WT mice. Thus, we conclude that ABIN-1 is a suppressor of innate immune response and the interaction of ABIN-1 with A20 controls innate immunity response through the NF-κB pathway and in both RIPK1 kinase activity-independent and dependent manner.

9.
Nat Rev Neurosci ; 20(1): 19-33, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30467385

RESUMO

Apoptosis is crucial for the normal development of the nervous system, whereas neurons in the adult CNS are relatively resistant to this form of cell death. However, under pathological conditions, upregulation of death receptor family ligands, such as tumour necrosis factor (TNF), can sensitize cells in the CNS to apoptosis and a form of regulated necrotic cell death known as necroptosis that is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like protein (MLKL). Necroptosis promotes further cell death and neuroinflammation in the pathogenesis of several neurodegenerative diseases, including multiple sclerosis, amyotrophic lateral sclerosis, Parkinson disease and Alzheimer disease. In this Review, we outline the evidence implicating necroptosis in these neurological diseases and suggest that targeting RIPK1 might help to inhibit multiple cell death pathways and ameliorate neuroinflammation.


Assuntos
Encéfalo/metabolismo , Inflamação/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Encéfalo/patologia , Humanos , Inflamação/patologia , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Fosforilação
10.
Front Mol Neurosci ; 11: 425, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30524233

RESUMO

Lithium is a widely used and effective treatment for individuals with psycho-neurological disorders, and it exhibits protective and regenerative properties in multiple brain injury animal models, but the clinical experience in young children is limited due to potential toxicity. As an interim analysis, this paper reports the safety/tolerability profiles of low-dose lithium treatment in children with intellectual disability (ID) and its possible beneficial effects. In a randomized, single-center clinical trial, 124 children with ID were given either oral lithium carbonate 6 mg/kg twice per day or the same dose of calcium carbonate as a placebo (n = 62/group) for 3 months. The safety of low-dose lithium treatment in children, and all the adverse events were monitored. The effects of low-dose lithium on cognition was evaluated by intelligence quotient (IQ), adaptive capacity was assessed by the Infant-Junior Middle School Students Social-Life Abilities Scale (IJMSSSLAS), and overall performance was evaluated according to the Clinical Global Impression-Improvement (CGI-I) scale. After 3 months of lithium treatment, 13/61 children (21.3%) presented with mild side effects, including 4 (6.6%) with gastrointestinal symptoms, 4 (6.6%) with neurological symptoms, 2 (3.3%) with polyuria, and 3 (4.9%) with other symptoms-one with hyperhidrosis, one with alopecia, and one with drooling. Four children in the lithium group had elevated blood thyroid stimulating hormone, which normalized spontaneously after lithium discontinuation. Both IQ and IJMSSSAS scores increased following 3 months of lithium treatment (F = 11.03, p = 0.002 and F = 7.80, p = 0.007, respectively), but such increases were not seen in the placebo group. CGI-I scores in the lithium group were 1.25 points lower (better) than in the placebo group (F = 82.66, p < 0.001) after 3 months of treatment. In summary, lithium treatment for 3 months had only mild and reversible side effects and had positive effects on cognition and overall performance in children with ID. Clinical Trial Registration: Chinese Clinical Trial Registry, ChiCTR-IPR-15007518.

12.
Proc Natl Acad Sci U S A ; 115(40): E9317-E9324, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30181285

RESUMO

Protooncogene c-MYC, a master transcription factor, is a major driver of human tumorigenesis. Development of pharmacological agents for inhibiting c-MYC as an anticancer therapy has been a longstanding but elusive goal in the cancer field. E3 ubiquitin ligase cIAP1 has been shown to mediate the activation of c-MYC by destabilizing MAD1, a key antagonist of c-MYC. Here we developed a high-throughput assay for cIAP1 ubiquitination and identified D19, a small-molecule inhibitor of E3 ligase activity of cIAP1. We show that D19 binds to the RING domain of cIAP1 and inhibits the E3 ligase activity of cIAP1 by interfering with the dynamics of its interaction with E2. Blocking cIAP1 with D19 antagonizes c-MYC by stabilizing MAD1 protein in cells. Furthermore, we show that D19 and an improved analog (D19-14) promote c-MYC degradation and inhibit the oncogenic function of c-MYC in cells and xenograft animal models. In contrast, we show that activating E3 ubiquitin ligase activity of cIAP1 by Smac mimetics destabilizes MAD1, the antagonist of MYC, and increases the protein levels of c-MYC. Our study provides an interesting example using chemical biological approaches for determining distinct biological consequences from inhibiting vs. activating an E3 ubiquitin ligase and suggests a potential broad therapeutic strategy for targeting c-MYC in cancer treatment by pharmacologically modulating cIAP1 E3 ligase activity.


Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ubiquitinação/efeitos dos fármacos , Animais , Antineoplásicos/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Cell ; 174(6): 1477-1491.e19, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30146158

RESUMO

Aging is a major risk factor for both genetic and sporadic neurodegenerative disorders. However, it is unclear how aging interacts with genetic predispositions to promote neurodegeneration. Here, we investigate how partial loss of function of TBK1, a major genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comorbidity, leads to age-dependent neurodegeneration. We show that TBK1 is an endogenous inhibitor of RIPK1 and the embryonic lethality of Tbk1-/- mice is dependent on RIPK1 kinase activity. In aging human brains, another endogenous RIPK1 inhibitor, TAK1, exhibits a marked decrease in expression. We show that in Tbk1+/- mice, the reduced myeloid TAK1 expression promotes all the key hallmarks of ALS/FTD, including neuroinflammation, TDP-43 aggregation, axonal degeneration, neuronal loss, and behavior deficits, which are blocked upon inhibition of RIPK1. Thus, aging facilitates RIPK1 activation by reducing TAK1 expression, which cooperates with genetic risk factors to promote the onset of ALS/FTD.


Assuntos
Apoptose , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Adulto , Idoso , Envelhecimento , Animais , Apoptose/efeitos dos fármacos , Axônios/metabolismo , Comportamento Animal , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Humanos , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Knockout , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Medula Espinal/metabolismo , Estaurosporina/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
14.
PLoS Biol ; 16(8): e2005756, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30157175

RESUMO

Necroptosis is a lytic programmed cell death mediated by the RIPK1-RIPK3-MLKL pathway. The loss of Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) expression and necroptotic potential have been previously reported in several cancer cell lines; however, the extent of this loss across cancer types, as well as its mutational drivers, were unknown. Here, we show that RIPK3 expression loss occurs progressively during tumor growth both in patient tumor biopsies and tumor xenograft models. Using a cell-based necroptosis sensitivity screen of 941 cancer cell lines, we find that escape from necroptosis is prevalent across cancer types, with an incidence rate of 83%. Genome-wide bioinformatics analysis of this differential necroptosis sensitivity data in the context of differential gene expression and mutation data across the cell lines identified various factors that correlate with resistance to necroptosis and loss of RIPK3 expression, including oncogenes BRAF and AXL. Inhibition of these oncogenes can rescue the RIPK3 expression loss and regain of necroptosis sensitivity. This genome-wide analysis also identifies that the loss of RIPK3 expression is the primary factor correlating with escape from necroptosis. Thus, we conclude that necroptosis resistance of cancer cells is common and is oncogene driven, suggesting that escape from necroptosis could be a potential hallmark of cancer, similar to escape from apoptosis.


Assuntos
Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Necrose/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Proc Natl Acad Sci U S A ; 115(26): E5944-E5953, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891719

RESUMO

Stimulation of cells with TNFα can promote distinct cell death pathways, including RIPK1-independent apoptosis, necroptosis, and RIPK1-dependent apoptosis (RDA)-the latter of which we still know little about. Here we show that RDA involves the rapid formation of a distinct detergent-insoluble, highly ubiquitinated, and activated RIPK1 pool, termed "iuRIPK1." iuRIPK1 forms after RIPK1 activation in TNF-receptor-associated complex I, and before cytosolic complex II formation and caspase activation. To identify regulators of iuRIPK1 formation and RIPK1 activation in RDA, we conducted a targeted siRNA screen of 1,288 genes. We found that NEK1, whose loss-of-function mutations have been identified in 3% of ALS patients, binds to activated RIPK1 and restricts RDA by negatively regulating formation of iuRIPK1, while LRRK2, a kinase implicated in Parkinson's disease, promotes RIPK1 activation and association with complex I in RDA. Further, the E3 ligases APC11 and c-Cbl promote RDA, and c-Cbl is recruited to complex I in RDA, where it promotes prodeath K63-ubiquitination of RIPK1 to lead to iuRIPK1 formation. Finally, we show that two different modes of necroptosis induction by TNFα exist which are differentially regulated by iuRIPK1 formation. Overall, this work reveals a distinct mechanism of RIPK1 activation that mediates the signaling mechanism of RDA as well as a type of necroptosis.


Assuntos
Apoptose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação , Animais , Linhagem Celular , Ativação Enzimática , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Necrose Tumoral alfa/genética
16.
Cell Death Dis ; 9(7): 732, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29955050

RESUMO

Parkin (Park2), a RING-between-RING-type E3 ubiquitin ligase, has been implicated in regulating NF-κB. Mutations in Parkin are associated with Parkinson's disease. Here we investigated the interaction of Parkin with Receptor-interacting protein kinase 1 (RIPK1) kinase, a key mediator of multiple signaling pathways activated by TNFR1 including NF-κB pathway. We report that Parkin interacts with RIPK1 and mediates K63 ubiquitination of RIPK1 on K376 in TNFR1-signaling pathway. The expression of Parkin promotes the recruitment of transforming growth factor ß (TGF-ß)-activated kinase 1 (TAK1), nuclear factor-κB (NF-κB) essential molecule (NEMO), Sharpin and A20 in complex I associated with TNFR1 upon TNFα stimulation. Ubiquitination of RIPK1 by Parkin increases the activation of NF-κB and mitogen-activated protein kinases (MAPKs) by promoting the phosphorylation of inhibitor of kappa B kinase (IKK)α/ß and IκBα and nuclear translocation of p65. Thus, we conclude that Parkin modulates the K63 ubiquitination status of RIPK1 to promote the activation of NF-κB and MAPKs.


Assuntos
NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Modelos Biológicos , Domínios Proteicos , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Fator de Necrose Tumoral alfa/farmacologia
17.
Cell Death Dis ; 9(5): 500, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29703889

RESUMO

Necroptosis, a form of regulated necrotic cell death, is mediated by receptor interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL). However, the mechanism by which necroptosis promotes inflammation is still unclear. Here we report that the expression of cytokines is robustly upregulated in a cell-autonomous manner during necroptosis induced by tumor necrosis factor alpha (TNFα). We demonstrate that TNFα-induced necroptosis leads to two waves of cytokine production. The first wave, more transient and weaker than the second, is in response to TNFα alone; whereas the second wave depends upon the necroptotic signaling. We show that necroptosis promotes the transcription of TNFα-target genes in a cell-intrinsic manner. The activation of both NF-κB and p38 by the necroptotic machinery, RIPK1, RIPK3, and MLKL, is involved in mediating the robust induction of cytokine expression in the second wave. In contrast, necroptosis induced by direct oligomerization of MLKL promotes cytokine production at much lower levels than that of necroptosis induced with TNFα. Thus, we conclude that TNFα-induced necroptosis signaling events mediated by RIPK1 and RIPK3 activation, in addition to the MLKL oligomerization, promotes the expression of cytokines involving multiple intracellular signaling mechanisms including NF-κB pathway and p38. These findings reveal that the necroptotic cell death machinery mounts an immune response by promoting cell-autonomous production of cytokines. Our study provides insights into the mechanism by which necroptosis promotes inflammation in human diseases.


Assuntos
Quimiocina CXCL1/genética , Regulação da Expressão Gênica/imunologia , Necrose/genética , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Linhagem Celular , Quimiocina CXCL1/agonistas , Quimiocina CXCL1/imunologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Células HEK293 , Células HT29 , Humanos , Camundongos , Necrose/imunologia , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteínas Quinases/imunologia , Multimerização Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Transdução de Sinais , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
18.
Genes Dev ; 32(5-6): 327-340, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29593066

RESUMO

Necroptosis, a form of regulated necrotic cell death mediated by RIPK1 (receptor-interacting protein kinase 1) kinase activity, RIPK3, and MLKL (mixed-lineage kinase domain-like pseudokinase), can be activated under apoptosis-deficient conditions. Modulating the activation of RIPK1 by ubiquitination and phosphorylation is critical to control both necroptosis and apoptosis. Mutant mice with kinase-dead RIPK1 or RIPK3 and MLKL deficiency show no detrimental phenotype in regard to development and adult homeostasis. However, necroptosis and apoptosis can be activated in response to various mutations that result in the abortion of the defective embryos and human inflammatory and neurodegenerative pathologies. RIPK1 inhibition represents a key therapeutic strategy for treatment of diseases where blocking both necroptosis and apoptosis can be beneficial.


Assuntos
Morte Celular/fisiologia , Doença , Crescimento e Desenvolvimento/fisiologia , Apoptose/genética , Crescimento e Desenvolvimento/genética , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
19.
Proc Natl Acad Sci U S A ; 115(9): E2001-E2009, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29440439

RESUMO

RIPK1 is a critical mediator of cell death and inflammation downstream of TNFR1 upon stimulation by TNFα, a potent proinflammatory cytokine involved in a multitude of human inflammatory and degenerative diseases. RIPK1 contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The kinase activity of RIPK1 promotes cell death and inflammation. Here, we investigated the involvement of RIPK1-DD in the regulation of RIPK1 kinase activity. We show that a charge-conserved mutation of a lysine located on the surface of DD (K599R in human RIPK1 or K584R in murine RIPK1) blocks RIPK1 activation in necroptosis and RIPK1-dependent apoptosis and the formation of complex II. Ripk1K584R/K584R knockin mutant cells are resistant to RIPK1 kinase-dependent apoptosis and necroptosis. The resistance of K584R cells, however, can be overcome by forced dimerization of RIPK1. Finally, we show that the K584R RIPK1 knockin mutation protects mice against TNFα-induced systematic inflammatory response syndrome. Our study demonstrates the role of RIPK1-DD in mediating RIPK1 dimerization and activation of its kinase activity during necroptosis and RIPK1-dependent apoptosis.


Assuntos
Apoptose , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Fator de Necrose Tumoral alfa/química , Motivos de Aminoácidos , Animais , Sobrevivência Celular , Ativação Enzimática , Éxons , Teste de Complementação Genética , Células HEK293 , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Necrose/genética , Fosforilação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
20.
Autophagy ; 14(1): 66-79, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29394115

RESUMO

OPTN (optineurin), a ubiquitin-binding scaffold protein, functions as an important macroautophagy/autophagy receptor in selective autophagy processes. Mutations in OPTN have been linked with human neurodegenerative diseases including ALS and glaucoma. However, the mechanistic basis underlying the recognition of ubiquitin by OPTN and its regulation by TBK1-mediated phosphorylation are still elusive. Here, we demonstrate that the UBAN domain of OPTN preferentially recognizes linear ubiquitin chain and forms an asymmetric 2:1 stoichiometry complex with the linear diubiquitin. In addition, our results provide new mechanistic insights into how phosphorylation of UBAN would regulate the ubiquitin-binding ability of OPTN and how disease-associated mutations in the OPTN UBAN domain disrupt its interaction with ubiquitin. Finally, we show that defects in ubiquitin-binding may affect the recruitment of OPTN to linear ubiquitin-decorated mutant Huntington protein aggregates. Taken together, our findings clarify the interaction mode between UBAN and linear ubiquitin chain in general, and expand our knowledge of the molecular mechanism of ubiquitin-decorated substrates recognition by OPTN as well as the pathogenesis of neurodegenerative diseases caused by OPTN mutations.


Assuntos
Autofagia , Doenças Neurodegenerativas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Ubiquitina/metabolismo , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Glaucoma/genética , Glaucoma/metabolismo , Células HeLa , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Fosforilação , Agregados Proteicos , Ligação Proteica , Fator de Transcrição TFIIIA/genética
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