RESUMO
Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.
Assuntos
COVID-19 , Classe Ib de Fosfatidilinositol 3-Quinase , Inflamação , SARS-CoV-2 , Animais , Humanos , Camundongos , Permeabilidade Capilar/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , COVID-19/patologia , Tratamento Farmacológico da COVID-19 , Síndrome da Liberação de Citocina/tratamento farmacológico , Inflamação/patologia , Pulmão/patologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , SARS-CoV-2/fisiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/patologiaRESUMO
DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.
Assuntos
Dano ao DNA , Histonas/metabolismo , Neoplasias/metabolismo , Ubiquitina/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Reparo do DNA , Histonas/fisiologia , Humanos , Camundongos , Modelos Biológicos , Fosforilação , Radiação Ionizante , Transdução de Sinais , Transfecção , Ubiquitina/metabolismoRESUMO
The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC.
Assuntos
Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Enterócitos/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Homeostase , Humanos , Íleo/patologia , Íleo/ultraestrutura , Integrases/metabolismo , Síndromes de Malabsorção/enzimologia , Síndromes de Malabsorção/patologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Organoides/metabolismo , Síndrome de EmaciaçãoRESUMO
Previous studies of the conditional ablation of TGF-ß activated kinase 1 (TAK1) in mice indicate that TAK1 has an obligatory role in the survival and/or development of hematopoietic stem cells, B cells, T cells, hepatocytes, intestinal epithelial cells, keratinocytes, and various tissues, primarily because of these cells' increased apoptotic sensitivity, and have implicated TAK1 as a critical regulator of the NF-κB and stress kinase pathways and thus a key intermediary in cellular survival. Contrary to this understanding of TAK1's role, we report a mouse model in which TAK1 deletion in the myeloid compartment that evoked a clonal myelomonocytic cell expansion, splenomegaly, multi-organ infiltration, genomic instability, and aggressive, fatal myelomonocytic leukemia. Unlike in previous reports, simultaneous deletion of TNF receptor 1 (TNFR1) failed to rescue this severe phenotype. We found that the features of the disease in our mouse model resemble those of human chronic myelomonocytic leukemia (CMML) in its transformation to acute myeloid leukemia (AML). Consequently, we found TAK1 deletion in 13 of 30 AML patients (43%), thus providing direct genetic evidence of TAK1's role in leukemogenesis.
Assuntos
Deleção de Genes , Leucemia Mielomonocítica Aguda/genética , MAP Quinase Quinase Quinases/genética , Animais , Citocinas/fisiologia , Citometria de Fluxo , Hibridização in Situ Fluorescente , Cariotipagem , Camundongos , Camundongos Knockout , Transdução de Sinais , Esplenomegalia/genéticaRESUMO
Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. Because Akt (also known as protein kinase B) resides primarily in the cytosol, it is not known how these signaling molecules are recruited to the plasma membrane and subsequently activated by growth factor stimuli. We found that the protein kinase Akt undergoes lysine-63 chain ubiquitination, which is important for Akt membrane localization and phosphorylation. TRAF6 was found to be a direct E3 ligase for Akt and was essential for Akt ubiquitination, membrane recruitment, and phosphorylation upon growth-factor stimulation. The human cancer-associated Akt mutant displayed an increase in Akt ubiquitination, in turn contributing to the enhancement of Akt membrane localization and phosphorylation. Thus, Akt ubiquitination is an important step for oncogenic Akt activation.
Assuntos
Membrana Celular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Interleucina-1beta/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/química , Fator 6 Associado a Receptor de TNF/genética , Transplante Heterólogo , UbiquitinaçãoRESUMO
Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-kappaB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.
Assuntos
Fator 6 Associado a Receptor de TNF/química , Enzimas de Conjugação de Ubiquitina/química , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Humanos , Conformação Proteica , Multimerização Proteica , Domínios RING Finger , Ubiquitinação , Dedos de ZincoRESUMO
TRAF6, a crucial adaptor molecule in innate and adaptive immunity, contains three distinct functional domains. The C-terminal TRAF domain facilitates oligomerization and sequence-specific interaction with receptors or other adaptor proteins. In conjunction with the dimeric E2 enzyme Ubc13-Uev1A, the N-terminal RING domain of TRAF6 functions as an E3 ubiquitin (Ub) ligase that facilitates its own site-specific ubiquitination through the generation of a Lys-63-linked poly-Ub chain. This modification does not cause its proteasomal degradation but rather serves as a scaffold to activate both the IKK and stress kinase pathways. Connecting the N-and C-terminal regions, the four internal zinc finger (ZF) motifs have yet to be functionally defined. In this study, we examined the role of the ZF domains in interleukin-1, lipopolysaccharide, and RANKL signaling by reconstitution of TRAF6-deficient cells with point mutations or deletions of these ZF motifs. Although ZF domains 2-4 are dispensable for activating IKK, p38, and JNK by interleukin-1 and lipopolysaccharide, the first ZF domain together with an intact RING domain of TRAF6 is essential for activating these pathways. Furthermore, TRAF6 autoubiquitination and its interaction with Ubc13 are dependent on ZF1 and an intact RING domain. Additionally, expression of TRAF6 lacking ZF2-4 in TRAF6-deficient monocytes rescues RANKL-mediated osteoclast differentiation and LPS-stimulated interleukin-6 production. These data provide evidence for the critical role of the Ub ligase activity of TRAF6, which is coordinated via the RING domain and ZF1 to supply the necessary elements in signaling by cytokines dependent upon TRAF6.
Assuntos
Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Ligante RANK/metabolismo , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Imunidade Inata/fisiologia , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Knockout , Monócitos/citologia , Monócitos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína/fisiologia , Ligante RANK/genética , Transdução de Sinais/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/fisiologia , Dedos de Zinco/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is a key mediator in proximal signaling of the interleukin-1/Toll-like receptor and the TNF receptor superfamily. Analysis of TRAF6-deficient mice revealed a fundamental role of TRAF6 in osteoclastogenesis; however, the molecular mechanism underlying TRAF6 signaling in this biological process is not understood. Recent biochemical evidence has indicated that TRAF6 possesses ubiquitin ligase activity that controls the activation of IKK and NF-kappaB. Because these studies are primarily based on cell-free systems, the role of the ubiquitin ligase activity of TRAF6 and its auto-ubiquitination to initiate the NF-kappaB pathway in vivo remain elusive. Here we show that an intact RING domain of TRAF6 in conjunction with the E2 enzyme Ubc13/Uev1A is necessary for Lys-63-linked auto-ubiquitination of TRAF6 and for its ability to activate IKK and NF-kappaB. Furthermore, a RING mutant of TRAF6 abolishes its ability to induce receptor activator of NF-kappaB-independent osteoclast differentiation and nuclear accumulation of the transcription factor NFATc1. Notably, we map the auto-ubiquitination site of TRAF6 to a single Lys residue, which if mutated renders TRAF6 unable to activate transforming growth factor-beta-activated kinase 1 and IKK and to cause spontaneous osteoclast differentiation. Additionally, we provide biochemical and in vivo evidence that TRAF6 serves as an E3 to directly ubiquitinate NEMO. Reconstituting TRAF6-deficent cells with various TRAF6 mutants, we clearly demonstrate the requirement for the TRAF6 RING domain and site-specific auto-ubiquitination of TRAF6 to activate IKK in response to interleukin-1. These data establish a signaling cascade in which regulated site-specific Lys-63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of IKK.
Assuntos
Quinase I-kappa B/metabolismo , Lisina/metabolismo , Fator 6 Associado a Receptor de TNF/química , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina/metabolismo , Animais , Catálise , Linhagem Celular , Ativação Enzimática , Humanos , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Estrutura Terciária de Proteína/genética , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/deficiência , Fator 6 Associado a Receptor de TNF/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/fisiologiaRESUMO
TRAF-interacting protein (TRIP) was initially identified as a TRAF1- and TRAF2-binding partner that inhibited NF-kappaB activation without a known mechanism. Inspection of the TRIP sequence revealed an N-terminal RING domain, which is found in many E3 ubiquitin (Ub) ligases. We show that TRIP is a RING-dependent Ub ligase that undergoes auto-ubiquitination and requires an intact RING domain. Both TRIP and its RING mutant interact with TRAF1, 2, 3, 5, and 6, but failed to interact with CYLD and NIK. Stable expression of TRIP or a RING mutant did not affect IKK activation induced by TNF or IL-1 and had no affect on TNF-induced apoptosis. Similarly, RANKL-induced signaling and osteoclastogenesis were not affected by TRIP or its RING mutant. Interestingly, TRIP expression was down regulated during the late stages of osteoclastogenesis. Taken together, our results demonstrate that TRIP is a novel RING-dependent Ub ligase and a binding partner for TRAFs.
Assuntos
Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Apoptose , Diferenciação Celular , Linhagem Celular , Enzima Desubiquitinante CYLD , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Quinase I-kappa B/metabolismo , Interleucina-1/farmacologia , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Tumor necrosis factor receptor-associated factor 6 (TRAF6), the crucial adaptor molecule of receptor activator of NF-kappaB (RANK), plays an essential role in governing the formation of multi-nucleated osteoclasts. TRAF6 is a RING-dependent ubiquitin (Ub) ligase that in conjunction with Ubc13/Uev1A catalyzes its own auto-ubiquitination via Lys63-linked poly-Ub chains. While the receptor-adaptor function of TRAF6 in RANK signaling is well understood, the significance of its Ub ligase activity in this process remains largely unknown. In this study, we show that retroviral expression of TRAF6, but not a RING mutant of TRAF6 was able to rescue TRAF6-deficient monocytes for the activation of IKK and osteoclast differentiation by RANKL. Furthermore, a catalytically inactive Ubc13 or stable knockdown of Ubc13 significantly prevents RANK-mediated TRAF6 ubiquitination and NF-kappaB and JNK activation. These data establish a signaling cascade in which regulated Lys63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of osteoclast differentiation.
Assuntos
Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Camundongos , Transdução de Sinais/fisiologiaRESUMO
Transforming growth factor beta-activated kinase 1 (TAK1), a member of the MAPKKK family, was initially described to play an essential role in the transforming growth factor beta-signaling pathway, but recent evidence has emerged implicating TAK1 in the interleukin (IL)-1 and tumor necrosis factor (TNF) pathways. Notably, two homologous proteins, TAB2 and TAB3, have been identified as adaptors linking TAK1 to the upstream adaptors TRAFs. However, it remains unclear whether the interaction between TAB2/TAB3 and TAK1 is necessary for its kinase activation and subsequent activation of the IKK and MAPK pathways. Here, we characterized the TAB2/TAB3-binding domain in TAK1 and further examined the requirement of this interaction for IL-1, TNF, and RANKL signaling. Through deletion mapping experiments, we demonstrated that the binding motif for TAB2/TAB3 is a non-contiguous region located within the last C-terminal 100 residues of TAK1. However, residues 479-553 of TAK1 appear to be necessary and sufficient for TAB2/TAB3 interaction. Conversely, residues 574-693 of TAB2 were shown to interact with TAK1. A green fluorescent protein fusion protein containing the last 100 residues of TAK1 (TAK1-C100) abolished the interaction of endogenous TAB2/TAB3 with TAK1, the phosphorylation of TAK1, and prevented the activation of IKK and MAPK induced by IL-1, TNF, and RANKL. Furthermore, TAK1-C100 blocked RANKL-induced nuclear accumulation of NFATc1 and consequently osteoclast differentiation consistent with the ability of a catalytically inactive TAK1 to block RANKL-mediated signaling. Significantly, our study provides evidence that the TAB2/TAB3 interaction with TAK1 is crucial for the activation of signaling cascades mediated by IL-1, TNF, and RANKL.