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1.
Proc Natl Acad Sci U S A ; 120(39): e2308079120, 2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37733743

RESUMO

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.


Assuntos
Apoptose , Neoplasias , Animais , Camundongos , Morte Celular , Citosol , Neoplasias/tratamento farmacológico , Neoplasias/genética , Ubiquitinação
2.
Cell Death Dis ; 13(9): 773, 2022 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-36071040

RESUMO

Activation of TNFR1 by TNFα induces the formation of a membrane-associated, intracellular complex termed complex I. Complex I orchestrates a complex pattern of modifications on key regulators of TNF signaling that collectively determines the cell fate by activating pro-survival or executing cell death programs. However, the regulatory mechanism of complex I in cell-fate decision is not fully understood. Here we identify protein phosphatase-6 (PP6) as a previously unidentified component of complex I. Loss of PP6 protects cells from TNFα-mediated cell death. The role of PP6 in regulating cell death requires its phosphatase activity and regulatory subunits. Further mechanistic studies show that PP6 modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL to promote RIPK1 activation and c-FLIPL degradation. We also show that melanoma-associated PP6 inactivating mutants offer resistance to cell death due to the loss of sensitivity to TNFα. Thus, our study provides a potential mechanism by which melanoma-related PP6 inactivating mutations promote cancer progression.


Assuntos
Melanoma , Fosfoproteínas Fosfatases , Fator de Necrose Tumoral alfa , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Morte Celular , Humanos , Fosfoproteínas Fosfatases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
3.
Cell Res ; 31(12): 1230-1243, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34663909

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the ongoing global pandemic that poses substantial challenges to public health worldwide. A subset of COVID-19 patients experience systemic inflammatory response, known as cytokine storm, which may lead to death. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important mediator of inflammation and cell death. Here, we examined the interaction of RIPK1-mediated innate immunity with SARS-CoV-2 infection. We found evidence of RIPK1 activation in human COVID-19 lung pathological samples, and cultured human lung organoids and ACE2 transgenic mice infected by SARS-CoV-2. Inhibition of RIPK1 using multiple small-molecule inhibitors reduced the viral load of SARS-CoV-2 in human lung organoids. Furthermore, therapeutic dosing of the RIPK1 inhibitor Nec-1s reduced mortality and lung viral load, and blocked the CNS manifestation of SARS-CoV-2 in ACE2 transgenic mice. Mechanistically, we found that the RNA-dependent RNA polymerase of SARS-CoV-2, NSP12, a highly conserved central component of coronaviral replication and transcription machinery, promoted the activation of RIPK1. Furthermore, NSP12 323L variant, encoded by the SARS-CoV-2 C14408T variant first detected in Lombardy, Italy, that carries a Pro323Leu amino acid substitution in NSP12, showed increased ability to activate RIPK1. Inhibition of RIPK1 downregulated the transcriptional induction of proinflammatory cytokines and host factors including ACE2 and EGFR that promote viral entry into cells. Our results suggest that SARS-CoV-2 may have an unexpected and unusual ability to hijack the RIPK1-mediated host defense response to promote its own propagation and that inhibition of RIPK1 may provide a therapeutic option for the treatment of COVID-19.


Assuntos
COVID-19/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/mortalidade , COVID-19/virologia , RNA-Polimerase RNA-Dependente de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Transgênicos , Mutação , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Taxa de Sobrevida , Transcriptoma/efeitos dos fármacos , Carga Viral/efeitos dos fármacos , Internalização do Vírus , Tratamento Farmacológico da COVID-19
4.
Nat Struct Mol Biol ; 27(4): 363-372, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231288

RESUMO

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.


Assuntos
Proteínas de Choque Térmico HSP27/química , Chaperonas Moleculares/química , Proteína FUS de Ligação a RNA/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/isolamento & purificação , Humanos , Extração Líquido-Líquido , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Fosforilação , Ligação Proteica/genética , Domínios Proteicos/genética , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Estresse Fisiológico/genética
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