Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
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.

2.
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
3.
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
5.
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
6.
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
7.
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
8.
Nat Cell Biol ; 20(1): 58-68, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29203883

RESUMO

Ubiquitylation of the TNFR1 signalling complex (TNF-RSC) controls the activation of RIPK1, a kinase critically involved in mediating multiple TNFα-activated deleterious events. However, the molecular mechanism that coordinates different types of ubiquitylation modification to regulate the activation of RIPK1 kinase remains unclear. Here, we show that ABIN-1/NAF-1, a ubiquitin-binding protein, is recruited rapidly into TNF-RSC in a manner dependent on the Met1-ubiquitylating complex LUBAC to regulate the recruitment of A20 to control Lys63 deubiquitylation of RIPK1. ABIN-1 deficiency reduces the recruitment of A20 and licenses cells to die through necroptosis by promoting Lys63 ubiquitylation and activation of RIPK1 with TNFα stimulation under conditions that would otherwise exclusively activate apoptosis in wild-type cells. Inhibition of RIPK1 kinase and RIPK3 deficiency block the embryonic lethality of Abin-1 -/- mice. We propose that ABIN-1 provides a critical link between Met1 ubiquitylation mediated by the LUBAC complex and Lys63 deubiquitylation by phospho-A20 to modulate the activation of RIPK1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fibroblastos/metabolismo , Fosfoproteínas/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Apoptose/genética , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Transformada , Fibroblastos/citologia , Regulação da Expressão Gênica , Genes Letais , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
9.
Genes Dev ; 31(11): 1162-1176, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28701375

RESUMO

Stimulation of cells with TNFα leads to the formation of the TNF-R1 signaling complex (TNF-RSC) to mediate downstream cellular fate decision. Activation of the TNF-RSC is modulated by different types of ubiquitination and may lead to cell death, including apoptosis and necroptosis, in both RIPK1-dependent and RIPK1-independent manners. Spata2 (spermatogenesis-associated 2) is an adaptor protein recruited into the TNF-RSC to modulate the interaction between the linear ubiquitin chain assembly complex (LUBAC) and the deubiquitinase CYLD (cylindromatosis). However, the mechanism by which Spata2 regulates the activation of RIPK1 is unclear. Here, we report that Spata2-deficient cells show resistance to RIPK1-dependent apoptosis and necroptosis and are also partially protected against RIPK1-independent apoptosis. Spata2 deficiency promotes M1 ubiquitination of RIPK1 to inhibit RIPK1 kinase activity. Furthermore, we provide biochemical evidence for the USP domain of CYLD and the PUB domain of the SPATA2 complex preferentially deubiquitinating the M1 ubiquitin chain in vitro. Spata2 deficiency also promotes the activation of MKK4 and JNK and cytokine production independently of RIPK1 kinase activity. Spata2 deficiency sensitizes mice to systemic inflammatory response syndrome (SIRS) induced by TNFα, which can be suppressed by RIPK1 inhibitor Nec-1s. Thus, Spata2 can regulate inflammatory response and cell death in both RIPK1-dependent and RIPK1-independent manners.


Assuntos
Proteínas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitinação/genética , Animais , Apoptose/genética , Células Cultivadas , Ativação Enzimática/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfotransferases/genética , Proteínas/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Síndrome de Resposta Inflamatória Sistêmica/enzimologia , Síndrome de Resposta Inflamatória Sistêmica/genética
10.
Cell ; 146(4): 607-20, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21854985

RESUMO

Previous experiments suggest a connection between the N-alpha-acetylation of proteins and sensitivity of cells to apoptotic signals. Here, we describe a biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the antiapoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We propose that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation.


Assuntos
Sobrevivência Celular , Proteínas/metabolismo , Proteína bcl-X/metabolismo , Acetilação , Animais , Apoptose , Caspase 2/metabolismo , Linhagem Celular , Embrião de Mamíferos/citologia , Técnicas de Inativação de Genes , Células HeLa , Humanos , Células Jurkat , Camundongos , Processamento de Proteína Pós-Traducional
11.
Cell Res ; 21(4): 588-99, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20877311

RESUMO

Using an image-based screen for small molecules that can affect Golgi morphology, we identify a small molecule, Sioc145, which can enlarge the Golgi compartments and promote protein secretion. More importantly, Sioc145 potentiates insulin secretion in a glucose-dependent manner. We show that Sioc145 selectively activates novel protein kinase Cs (nPKCs; δ and ɛ) but not conventional PKCs (cPKCs; α, ßI and ßII) in INS-1E insulinoma cells. In contrast, PMA, a non-selective activator of cPKCs and nPKCs, promotes insulin secretion independent of glucose concentrations. Furthermore, we demonstrate that Sioc145 and PMA show differential abilities in depolarizing the cell membrane, and suggest that Sioc145 promotes insulin secretion in the amplifying pathway downstream of K(ATP) channels. In pancreatic islets, the treatment with Sioc145 enhances the second phase of insulin secretion. Increased insulin granules close to the plasma membrane are observed after Sioc145 treatment. Finally, the administration of Sioc145 to diabetic GK rats increases their serum insulin levels and improves glucose tolerance. Collectively, our studies identify Sioc145 as a novel glucose-dependent insulinotropic compound via selectively activating nPKCs.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Proteína Quinase C/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Imunofluorescência , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Insulina/sangue , Secreção de Insulina , Insulinoma/metabolismo , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/metabolismo , Microscopia Eletrônica , Canais de Potássio/fisiologia , Ratos
12.
J Biol Chem ; 283(45): 31087-96, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-18799457

RESUMO

An image-based phenotypic screen was developed to identify small molecule regulators of intracellular traffic. Using this screen we found that AG1478, a previously known inhibitor of epidermal growth factor receptor, had epidermal growth factor receptor-independent activity in inducing the disassembly of the Golgi in human cells. Similar to brefeldin A (BFA), a known disrupter of the Golgi, AG1478 inhibits the activity of small GTPase ADP-ribosylation factor. Unlike BFA, AG1478 exhibits low cytotoxicity and selectively targets the cis-Golgi without affecting endosomal compartment. We show that AG1478 inhibits GBF1, a large nucleotide exchange factor for the ADP-ribosylation factor, in a Sec7 domain-dependent manner and mimics the phenotype of a GBF1 mutant that has an inactive mutation. The treatment with AG1478 leads to the recruitment of GBF1 to the vesicular-tubular clusters adjacent to the endoplasmic reticulum exit sites, a step only transiently observed previously in the presence of BFA. We propose that the treatment with AG1478 delineates a membrane trafficking intermediate step that depends upon the Sec7 domain.


Assuntos
Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Membranas Intracelulares/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Tirfostinas/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Brefeldina A/farmacologia , Linhagem Celular , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Mutação , Estrutura Terciária de Proteína/genética , Quinazolinas
13.
Proc Natl Acad Sci U S A ; 104(48): 19023-8, 2007 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-18024584

RESUMO

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.


Assuntos
Autofagia/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Fluorescência Verde/análise , Proteínas Associadas aos Microtúbulos/análise , Fagossomos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/instrumentação , Fluspirileno/farmacologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Membranas Intracelulares/química , Loperamida/farmacologia , Micotoxinas/farmacologia , Peptídeos/metabolismo , Fagossomos/química , Fosfatos de Fosfatidilinositol/metabolismo , Pimozida/farmacologia , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão/análise , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Trifluoperazina/farmacologia , Dedos de Zinco/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA