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1.
Molecules ; 26(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34361750

RESUMO

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.


Assuntos
Acacia/química , Antineoplásicos Fitogênicos/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Triterpenos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Apoptose/genética , Sítios de Ligação , Caseína Quinase Iépsilon/antagonistas & inibidores , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Células K562 , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Quinases Relacionadas a NIMA/antagonistas & inibidores , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Casca de Planta/química , Extratos Vegetais/química , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais , Triterpenos/química , Triterpenos/isolamento & purificação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
2.
Nat Commun ; 12(1): 4536, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315872

RESUMO

Despite the substantial impact of post-translational modifications on programmed cell death 1 ligand 1 (PD-L1), its importance in therapeutic resistance in pancreatic cancer remains poorly defined. Here, we demonstrate that never in mitosis gene A-related kinase 2 (NEK2) phosphorylates PD-L1 to maintain its stability, causing PD-L1-targeted pancreatic cancer immunotherapy to have poor efficacy. We identify NEK2 as a prognostic factor in immunologically "hot" pancreatic cancer, involved in the onset and development of pancreatic tumors in an immune-dependent manner. NEK2 deficiency results in the suppression of PD-L1 expression and enhancement of lymphocyte infiltration. A NEK binding motif (F/LXXS/T) is identified in the glycosylation-rich region of PD-L1. NEK2 interacts with PD-L1, phosphorylating the T194/T210 residues and preventing ubiquitin-proteasome pathway-mediated degradation of PD-L1 in ER lumen. NEK2 inhibition thereby sensitizes PD-L1 blockade, synergically enhancing the anti-pancreatic cancer immune response. Together, the present study proposes a promising strategy for improving the effectiveness of pancreatic cancer immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Imunidade , Quinases Relacionadas a NIMA/antagonistas & inibidores , Neoplasias Pancreáticas/imunologia , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Modelos Biológicos , Quinases Relacionadas a NIMA/deficiência , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação , Fosfosserina/metabolismo , Prognóstico , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Ubiquitinação
3.
Theranostics ; 11(8): 3898-3915, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33664869

RESUMO

Prolactin binding to the prolactin receptor exerts pleiotropic biological effects in vertebrates. The prolactin receptor (PRLR) has multiple isoforms due to alternative splicing. The biological roles and related signaling of the long isoform (PRLR-LF) have been fully elucidated. However, little is known about the short isoform (PRLR-SF), particularly in cancer development and metabolic reprogramming, a core hallmark of cancer. Here, we reveal the role and underlying mechanism of PRLR-SF in pancreatic ductal adenocarcinoma (PDAC). Methods: A human PDAC tissue array was used to investigate the clinical relevance of PRLR in PDAC. The in vivo implications of PRLR-SF in PDAC were examined in a subcutaneous xenograft model and an orthotopic xenograft model. Immunohistochemistry was performed on tumor tissue obtained from genetically engineered KPC (KrasG12D/+; Trp53R172H/+; Pdx1-Cre) mice with spontaneous tumors. 13C-labeled metabolite measures, LC-MS, EdU incorporation assays and seahorse analyses were used to identify the effects of PRLR-SF on the pentose phosphate pathway and glycolysis. We identified the molecular mechanisms by immunofluorescence, coimmunoprecipitation, proximity ligation assays, chromatin immunoprecipitation and promoter luciferase activity. Public databases (TCGA, GEO and GTEx) were used to analyze the expression and survival correlations of the related genes. Results: We demonstrated that PRLR-SF is predominantly expressed in spontaneously forming pancreatic tumors of genetically engineered KPC mice and human PDAC cell lines. PRLR-SF inhibits the proliferation of PDAC cells (AsPC-1 and BxPC-3) in vitro and tumor growth in vivo. We showed that PRLR-SF reduces the expression of genes in the pentose phosphate pathway (PPP) and nucleotide biosynthesis by activating Hippo signaling. TEAD1, a downstream transcription factor of Hippo signaling, directly regulates the expression of G6PD and TKT, which are PPP rate-limiting enzymes. Moreover, NEK9 directly interacts with PRLR-SF and is the intermediator between PRLR and the Hippo pathway. The PRLR expression level is negatively correlated with overall survival and TNM stage in PDAC patients. Additionally, pregnancy and lactation increase the ratio of PRLR-SF:PRLR-LF in the pancreas of wild-type mice and subcutaneous PDAC xenograft tumors. Conclusion: Our characterization of the relationship between PRLR-SF signaling, the NEK9-Hippo pathway, PPP and nucleotide synthesis explains a mechanism for the correlation between PRLR-SF and metabolic reprogramming in PDAC progression. Strategies to alter this pathway might be developed for the treatment or prevention of pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores da Prolactina/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Glucosefosfato Desidrogenase/genética , Xenoenxertos , Humanos , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Nucleotídeos/biossíntese , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Via de Pentose Fosfato , Medicina de Precisão , Prognóstico , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Prolactina/química , Receptores da Prolactina/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcetolase/genética
4.
Cancer Sci ; 112(5): 1695-1706, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33605517

RESUMO

Wnt/ß-catenin signaling is indispensable for many biological processes, including embryonic development, cell cycle, inflammation, and carcinogenesis. Aberrant activation of the Wnt/ß-catenin signaling can promote tumorigenicity and enhance metastatic potential in hepatocellular carcinoma (HCC). Targeting this pathway is a new opportunity for precise medicine for HCC. However, inhibiting Wnt/ß-catenin signaling alone is unlikely to significantly improve HCC patient outcome due to the lack of specific inhibitors and the complexity of this pathway. Combination with other therapies will be an important next step in improving the efficacy of Wnt/ß-catenin signaling inhibitors. Protein kinases play a key and evolutionarily conserved role in the Wnt/ß-catenin signaling and have become one of the most important drug targets in cancer. Targeting Wnt/ß-catenin signaling and its regulatory kinase together will be a promising HCC management strategy. In this review, we summarize the kinases that modulate the Wnt/ß-catenin signaling in HCC and briefly discuss their molecular mechanisms. Furthermore, we list some small molecules that target the kinases and may inhibit Wnt/ß-catenin signaling, to offer new perspectives for preclinical and clinical HCC studies.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Quinases/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/antagonistas & inibidores , Complexo de Sinalização da Axina/metabolismo , Proteína Quinase CDC2/metabolismo , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/terapia , Terapia Combinada/métodos , Creatina Quinase/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Receptores ErbB/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/terapia , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Medicina de Precisão , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Quinases Ativadas por p21/metabolismo , Quinases da Família src/metabolismo
5.
Theranostics ; 11(5): 2460-2474, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33500736

RESUMO

Rationale: Inflammatory stimuli from the tumor microenvironment play important roles in cancer progression. However, the mechanism of promotion of cancer metastasis by inflammation in gastric cancer (GC) is poorly understood. Methods: The roles of NEK9 were validated via loss-of-function and gain-of-function experiments in vitro and in an animal model of metastasis. Cytoskeletal reorganization-associated molecules were detected by GST pull-down. The regulation of ARHGEF2 by NEK9 was investigated by phosphoproteomics analysis, immunoprecipitation (IP) and in vitro kinase assay. The transcriptional regulation of miR-520f-3p was studied using luciferase reporter and chromatin immunoprecipitation (ChIP). The expression of these proteins in GC tissues was examined by immunohistochemistry. Results: NEK9 directly regulates cell motility and RhoA activation in GC. The phosphorylation of ARHGEF2 by NEK9 is the key step of this process. NEK9 is a direct target of miR-520f-3p, which is transcriptionally suppressed by IL-6-mediated activation of STAT3. A decrease in miR-520f-3p leads to the amplification of IL-6/STAT3 by targeting GP130. A simultaneous elevation of the levels of NEK9, GP130 and p-STAT3 was confirmed in the lymph nodes and distant metastases. An increase in NEK9, GP130 and STAT3 is associated with reduced overall survival of GC patients. Conclusion: This study demonstrates that activation of STAT3 by IL-6 transcriptionally suppresses miR-520f-3p and diminishes the inhibitory effects of miR-520f-3p on NEK9 and GP130. An increase in GP130 enhances this signaling, and NEK9 directly influences cell motility and RhoA activation by targeting the phosphorylation of ARHGEF2. Targeting the IL-6-STAT3-NEK9 pathway may be a new strategy for GC treatment.


Assuntos
Interleucina-6/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Quinases Relacionadas a NIMA/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Fator de Transcrição STAT3/metabolismo , Neoplasias Gástricas/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-6/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Quinases Relacionadas a NIMA/genética , Fosforilação , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fator de Transcrição STAT3/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorais Cultivadas , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Int Immunopharmacol ; 91: 107298, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33388733

RESUMO

BACKGROUND: Oridonin (Ori) has multiple biological properties, especially anti-inflammatory. However, its effects on chronic unpredictable mild stress (CUMS)-induced insulin resistance are still unclear. In this study, we explored the regulatory role of Ori in CUMS-triggered insulin resistance, and the underlying molecular mechanisms; Methods: SD rats were subjected to CUMS for 4 weeks, some of which were injected with Ori or fluoxetine (FLX) in durations of CUMS. After CUMS procedure, the behavioral and metabolic tests were performed. Elisa, immunofluorescence and western blotting were used to determine the inflammatory response and NLRP3 inflammasome activation. We investigated the interaction between NLRP3 and NEK7 using immunoprecipitation. Finally, we detected the proinflammatory cytokines in Lipopolysaccharide (LPS)-activated RAW264.7 cells treated with Ori; RESULTS: In this study, we found that chronic stress resulted in depressive-like behavior comorbid with insulin resistance. Ori was discovered to ameliorate insulin resistance as well as insulin signaling disturbance in the hippocampus. In addition, CUMS caused the infiltration of macrophages into the islets. And IL-1ß, IL-18 and caspase-1 were elevated in pancreases of CUMS rats, which could also be reversed by Ori treatment via reducing the interaction between NLRP3 and NEK7. Furthermore, Ori dose-dependently inhibited the levels of IL-1ß and IL-18 in LPS-activated RAW264.7 cells; CONCLUSIONS: All these results supported our hypothesis that Ori possesses potent anti-insulin resistant actions, which is partially correlated with inhibiting infiltration of macrophages into the islets and NLRP3 activation induced by CUMS. Therefore, our results highlighted the protective role of Ori against CUMS-elicited insulin resistance.


Assuntos
Anti-Inflamatórios/farmacologia , Diterpenos do Tipo Caurano/farmacologia , Inflamassomos/metabolismo , Resistência à Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estresse Psicológico/tratamento farmacológico , Animais , Comportamento Animal/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Doença Crônica , Citocinas/sangue , Modelos Animais de Doenças , Inflamassomos/imunologia , Mediadores da Inflamação/sangue , Insulina/sangue , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Quinases Relacionadas a NIMA/metabolismo , Células RAW 264.7 , Ratos Sprague-Dawley , Estresse Psicológico/imunologia , Estresse Psicológico/metabolismo
7.
Int Immunopharmacol ; 92: 107358, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33508701

RESUMO

INF39 is a nontoxic, irreversible, acrylate-based NLRP3 inhibitor and a further optimization of ethyl 2-((2-chlorophenyl) hydroxyl) methyl) acrylate (INF4E). However, the detail mechanism and the direct target of its anti-inflammatory activity is not clear. Here, we show that INF39 is a specific inhibitor for NLRP3 inflammasome activation. INF39 specifically suppresses NLRP3 activation but not the NLRC4 or AIM2 inflammasomes. INF39 has no effect on K+ efflux, ROS generation or mitochondrial membrane potential, which are the upstream events of NLRP3 inflammasome activation. In addition, INF39 has no direct inhibitory effect on GSDMD, which is the downstream event of inflammasomes. More importantly, INF39 inhibits the interaction of NEK7-NLRP3, and subsequently inhibits interaction of NLRP3-NLRP3, NLRP3-ASC, ASC oligomerization and speckle formation. Altogether, our study unveils a deeper anti-inflammatory mechanism for INF39 and suggests it could serve as a lead for developing novel therapeutics combating NLRP3-driven diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Inflamassomos/antagonistas & inibidores , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Metacrilatos/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular , Humanos , Inflamassomos/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Camundongos , Quinases Relacionadas a NIMA/metabolismo
8.
J Med Chem ; 64(1): 768-781, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33440945

RESUMO

Berberine (BBR), a traditional Chinese medicine, has therapeutic effects on a variety of inflammation-related diseases, but its direct proteomic targets remain unknown. Using activity-based protein profiling, we first demonstrated that BBR directly targets the NEK7 protein via the hydrogen bond between the 2,3-methylenedioxy and 121-arginine (R121) residues. The fact that R121 is located precisely within the key domain involved in the NEK7-NLRP3 interaction allows BBR to specifically block the NEK7-NLRP3 interaction and successively inhibit IL-1ß release, independent of the NF-κB and TLR4 signaling pathways. Moreover, BBR displays in vivo anti-inflammatory efficacy in a NEK7-dependent manner. Therefore, we consider NEK7 to be a key target of BBR in the treatment of NLRP3-related inflammatory diseases, and the development of novel NEK7-NLRP3 interaction inhibitors might be easily achieved using NEK7 as a target.


Assuntos
Anti-Inflamatórios/química , Berberina/química , Quinases Relacionadas a NIMA/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Berberina/metabolismo , Berberina/farmacologia , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Quinases Relacionadas a NIMA/antagonistas & inibidores , Quinases Relacionadas a NIMA/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
9.
Exp Cell Res ; 398(2): 112418, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33309808

RESUMO

BACKGROUND: NIMA-related kinase-7 (NEK7) is a serine/threonine kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined. METHODS: In this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 µg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP). RESULTS: Here, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord. CONCLUSION: NEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.


Assuntos
Inflamassomos/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Traumatismos da Medula Espinal/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Traumatismos da Medula Espinal/cirurgia
10.
Molecules ; 25(23)2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33255820

RESUMO

Inflammasomes are protein complexes which are important in several inflammatory diseases. Inflammasomes form part of the innate immune system that triggers the activation of inflammatory cytokines interleukin (IL)-1ß and IL-18. The inflammasome most studied in sterile inflammation and non-communicable disease is the NLRP3 inflammasome. Upon activation by diverse pathogen or disease associated signals, NLRP3 nucleates the oligomerization of an adaptor protein ASC forming a platform (the inflammasome) for the recruitment and activation of the protease caspase-1. Active caspase-1 catalyzes the processing and release of IL-1ß and IL-18, and via cleavage of the pore forming protein gasdermin D can drive pyroptotic cell death. This review focuses on the structural basis and mechanism for NLRP3 inflammasome signaling in the context of drug design, providing chemical structures, activities, and clinical potential of direct inflammasome inhibitors. A cryo-EM structure of NLRP3 bound to NEK7 protein provides structural insight and aids in the discovery of novel NLRP3 inhibitors utilizing ligand-based or structure-based approaches.


Assuntos
Descoberta de Drogas , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Ensaios Clínicos como Assunto , Desenho de Fármacos , Desenvolvimento de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunidade Inata , Inflamação/etiologia , Inflamação/metabolismo , Modelos Moleculares , Quinases Relacionadas a NIMA/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Ligação Proteica , Relação Estrutura-Atividade
11.
Cell Death Dis ; 11(11): 1005, 2020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-33230144

RESUMO

Defective left-right (LR) organization involving abnormalities in cilia ultrastructure causes laterality disorders including situs inversus (SI) and heterotaxy (Htx) with the prevalence approximately 1/10,000 births. In this study, we describe two unrelated family trios with abnormal cardiac LR patterning. Through whole-exome sequencing (WES), we identified compound heterozygous mutations (c.805-1G >C; p. Ile269GlnfsTer8/c.1117dupA; p.Thr373AsnfsTer19) (c.29T>C; p.Ile10Thr/c.356A>G; p.His119Arg) of NEK3, encoding a NIMA (never in mitosis A)-related kinase, in two affected individuals, respectively. Protein levels of NEK3 were abrogated in Patient-1 with biallelic loss-of function (LoF) NEK3 mutations that causes premature stop codon. Subsequence transcriptome analysis revealed that NNMT (nicotinamide N-methyltransferase) and SIRT2 (sirtuin2) was upregulated by NEK3 knockdown in human retinal pigment epithelial (RPE) cells in vitro, which associates α-tubulin deacetylation by western blot and immunofluorescence. Transmission electron microscopy (TEM) analysis further identified defective ciliary ultrastructure in Patient-1. Furthermore, inner ring components of nuclear pore complex (NPC) including nucleoporin (NUP)205, NUP188, and NUP155 were significantly downregulated in NEK3-silenced cells. In conclusion, we identified biallelic mutations of NEK3 predispose individual to abnormal cardiac left-right patterning via SIRT2-mediated α-tubulin deacetylation and downregulation of inner ring nucleoporins. Our study suggested that NEK3 could be a candidate gene for human ciliopathies.


Assuntos
Quinases Relacionadas a NIMA/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Adulto , Criança , Regulação para Baixo , Feminino , Humanos , Masculino , Mutação , Tubulina (Proteína)/metabolismo
12.
BMC Med Genomics ; 13(1): 158, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33109182

RESUMO

BACKGROUND: NEK2 has an established involvement in hepatocellular carcinoma (HCC) but the roles of NEK2 and its interacting proteins in HCC have not been systematically explored. METHODS: This study examined NEK2 and its interacting proteins in HCC based on multiple databases. RESULTS: NEK2 mRNA was highly expressed in HCC tissues compared with normal liver tissues. The survival of HCC patients with high NEK2 mRNA expression was shorter than those with low expression. MAD1L1, CEP250, MAPK1, NDC80, PPP1CA, PPP1R2 and NEK11 were the interacting proteins of NEK2. Among them, NDC80 and CEP250 were the key interacting proteins of NEK2. Mitotic prometaphase may be the key pathway that NEK2 and its interacting proteins contributed to HCC pathogenesis. NEK2, NDC80 and CEP250 mRNAs were highly expressed in HCC tissues compared with normal liver tissues. The mRNA levels of NEK2 were positively correlated with those of NDC80 or CEP250. Univariate regression showed that NEK2, NDC80 and CEP250 mRNA expressions were significantly associated with HCC patients' survival. Multivariate regression showed that NDC80 mRNA expression was an independent predictor for HCC patients' survival. Methylations and genetic alterations of NEK2, NDC80 and CEP250 were observed in HCC samples. The alterations of NEK2, NDC80 and CEP250 genes were co-occurrence. Patients with high mRNA expression and genetic alterations of NEK2, NDC80 and CEP250 had poor prognosis. CONCLUSIONS: NEK2 and its interacting proteins NDC80 and CEP250 play important roles in HCC development and progression and thus may be potentially used as biomarkers and therapeutic targets of HCC.


Assuntos
Autoantígenos/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Domínios e Motivos de Interação entre Proteínas , Autoantígenos/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Quinases Relacionadas a NIMA/genética , Prognóstico , Taxa de Sobrevida
13.
Oncogene ; 39(44): 6816-6840, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32978522

RESUMO

Progression through mitosis is balanced by the timely regulation of phosphorylation and dephosphorylation events ensuring the correct segregation of chromosomes before cytokinesis. This balance is regulated by the opposing actions of CDK1 and PP2A, as well as the Greatwall kinase/MASTL. MASTL is commonly overexpressed in cancer, which makes it a potential therapeutic anticancer target. Loss of Mastl induces multiple chromosomal errors that lead to the accumulation of micronuclei and multilobulated cells in mitosis. Our analyses revealed that loss of Mastl leads to chromosome breaks and abnormalities impairing correct segregation. Phospho-proteomic data for Mastl knockout cells revealed alterations in proteins implicated in multiple processes during mitosis including double-strand DNA damage repair. In silico prediction of the kinases with affected activity unveiled NEK2 to be regulated in the absence of Mastl. We uncovered that, RAD51AP1, involved in regulation of homologous recombination, is phosphorylated by NEK2 and CDK1 but also efficiently dephosphorylated by PP2A/B55. Our results suggest that MastlKO disturbs the equilibrium of the mitotic phosphoproteome that leads to the disruption of DNA damage repair and triggers an accumulation of chromosome breaks even in noncancerous cells.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Quebra Cromossômica , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Quinases Relacionadas a NIMA/metabolismo , Fosforilação/genética , Cultura Primária de Células , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteômica , Proteínas de Ligação a RNA/metabolismo
14.
Bioorg Med Chem ; 28(23): 115775, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32992252

RESUMO

Never in mitosis (NIMA) related kinase 2 (Nek2) is involved in multiple cellular processes such as cell cycle checkpoint regulation, cell division, DNA damage response and cell apoptosis. Nek2 has been reported to be overexpressed in various tumors and correlated with poor prognosis. Herein, a series of imidazo[1,2-a] pyridines Nek2 inhibitors were designed, synthesized, and their biological activities were investigated. Besides, structure activity relationship analysis of these compounds were performed in the MGC-803 cell. The screening results are promising, and compound 28e shows good proliferation inhibitory activity with an IC50 of 38 nM. The results would be helpful to design and develop more effective Nek2 inhibitors for the treatment of gastric cancer.


Assuntos
Desenho de Fármacos , Quinases Relacionadas a NIMA/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Piridinas/química , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Quinases Relacionadas a NIMA/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/metabolismo , Piridinas/farmacologia , Relação Estrutura-Atividade
15.
J Exp Clin Cancer Res ; 39(1): 183, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32907622

RESUMO

BACKGROUND: NEK2, a serine/threonine kinase involved in mitosis, has been found to function in chromosome instability, tumor progression and metastasis, but its role in cervical cancer radioresistance remains unknown. METHODS: We detected the protein levels of NEK2 in cervical carcinoma tissues and paired paracarcinoma tissues by immunohistochemistry. The roles of NEK2 in oncogenesis were examined using cell growth and colony formation assays, EdU assay, apoptosis assay as well as in vivo mouse model. γ-H2AX and Rad51 foci formation, neutral comet assay and clonogenic cell survival assay were applied to determine the radiosensitivity of cervical cancer cells. RNA-seq was performed to identify the downstream effector of NEK2. The gene expression levels were measured by Real-time PCR. RESULTS: We report that NEK2 protein level is overexpressed and correlated with the tumor stage and lymph node metastasis in cervical cancer tissues. Furthermore, we provided evidence that depletion of NEK2 impairs oncogenesis and enhances radiosensitivity in cervical cancer. Using RNA sequencing, we identify Wnt1 as a key downstream effector of NEK2. Knockdown of NEK2 downregulates the mRNA and protein levels of Wnt1, thereby inhibiting the activation of the Wnt/ß-catenin signaling pathway. More importantly, the observed consequences induced by NEK2 depletion in cervical cancer cells can be partially rescued by Wnt1 overexpression. CONCLUSIONS: Our results demonstrate that NEK2 activates the Wnt/ß-catenin signaling pathway via Wnt1 to drive oncogenesis and radioresistance in cervical cancer, indicating that NEK2 may be a promising target for the radiosensitization of cervical cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Quinases Relacionadas a NIMA/metabolismo , Tolerância a Radiação , Neoplasias do Colo do Útero/patologia , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Humanos , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Quinases Relacionadas a NIMA/genética , Prognóstico , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/radioterapia , Proteína Wnt1/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
16.
Mol Cell Biochem ; 475(1-2): 15-25, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32761510

RESUMO

Activating mutations within the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR) gene are observed in 10 ~ 30% of the patients diagnosed with non-small cell lung cancer (NSCLC), and are causally related to NSCLC initiation and progression. Treatments with tyrosine kinase inhibitors (TKIs) targeting EGFR significantly improve the outcome of NSCLC patients with EGFR mutation, but are often associated with drug resistance, which is the main cause of treatment failure and cancer relapse. In the present study, by screening the transcriptome of NSCLC patients, we found that EGFR activation is highly correlated with the up-regulation of mitotic regulator, never in mitosis gene A-related kinase 2 (NEK2). NEK2 overexpression is associated with the poor survival of EGFR-mutant patients but not the wild-type patients. Further functional validation revealed that EGFR mutation induces NEK2 expression by activating ERK signaling pathway. Elevated NEK2 level promotes the rapid cell cycle progression and favors the rapid proliferation of EGFR-mutant NSCLC cells. Of note, NEK2 overexpression also impairs the efficacy of TKI treatment via inhibiting apoptosis, while depleting NEK2 suppresses cell growth and restored the sensitivity of TKI in NSCLC cells. Taken together, our study revealed that NEK2 is an oncogene regulated by EGFR mutation and is involved in disease progression and treatment response in NSCLC with EGFR mutation. These findings will pave the road for optimizing personalized treatment strategies to overcome drug resistance and improve the prognosis of lung cancer patients with EGFR mutation.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Mutação , Quinases Relacionadas a NIMA/biossíntese , Recidiva Local de Neoplasia/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Bases de Dados Genéticas , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Prognóstico , Transdução de Sinais , Taxa de Sobrevida
17.
J Cell Mol Med ; 24(13): 7353-7369, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32558224

RESUMO

Emerging evidence has reported that dysregulation of microRNAs (miRNAs) participated in the development of diverse types of cancers. Our initial microarray-based analysis identified differentially expressed NEK2 related to breast cancer and predicted the regulatory microRNA-128-3p (miR-128-3p). Herein, this study aimed to characterize the tumour-suppressive role of miR-128-3p in regulating the biological characteristics of breast cancer stem cells (BCSCs). CD44+ CD24-/low cells were selected for subsequent experiments. After verification of the target relationship between miR-128-3p and NEK2, the relationship among miR-128-3p, NEK2 and BCSCs was further investigated with the involvement of the Wnt signalling pathway. The regulatory effects of miR-128-3p on proliferation, migration, invasion and self-renewal in vitro as well as tumorigenicity in vivo of BCSCs were examined via gain- and loss-of-function approaches. Highly expressed NEK2 was found in breast cancer based on GSE61304 expression profile. Breast cancer stem cells and breast cancer cells showed a down-regulation of miR-128-3p. Overexpression of miR-128-3p was found to inhibit proliferation, migration, invasion, self-renewal in vitro and tumorigenicity in vivo of BCSCs, which was further validated to be achieved through inhibition of Wnt signalling pathway by down-regulating NEK2. In summary, this study indicates that miR-128-3p inhibits the stem-like cell features of BCSCs via inhibition of the Wnt signalling pathway by down-regulating NEK2, which provides a new target for breast cancer treatment.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Quinases Relacionadas a NIMA/genética , Células-Tronco Neoplásicas/patologia , Via de Sinalização Wnt , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Autorrenovação Celular/genética , Feminino , Inativação Gênica , Humanos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Quinases Relacionadas a NIMA/metabolismo , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Regulação para Cima/genética
18.
Biochem Pharmacol ; 177: 113998, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32353421

RESUMO

Disordered immune regulation and persistent inflammatory damage are the key mechanisms of ventilator-induced lung injury (VILI). NLR family pyrin domain containing 3 (NLRP3) inflammasome activation causes VILI by mediating the formation of inflammatory mediators and infiltration of inflammatory cells, increasing pulmonary capillary membrane permeability, which leads to pulmonary edema and lung tissue damage. What mediates activation of NLRP3 inflammasome in VILI? In this study, we constructed an in vitro cyclic stretch (CS)-stimulated mouse lung epithelial (MLE-12) cell model that was transfected with NIMA-related kinase 7 (NEK7) small interfering RNA (siRNA) or scramble siRNA (sc siRNA) and pretreated with or without glibenclamide (glb). We also established a VILI mouse model, which was pretreated with glibenclamide or oridonin (Ori). Our goal was to investigate the regulatory effects of NEK7 on NLRP3 inflammasome activation and the anti-inflammatory effects of glibenclamide and oridonin on VILI. Mechanical stretch exaggerated the interaction between NEK7 and NLRP3, leading to assembly and activation of NLRP3 inflammasome downstream of potassium efflux. NEK7 depletion and treatment with glibenclamide or oridonin exerted anti-inflammatory effects that alleviated VILI by blocking the interaction between NEK7 and NLRP3, inhibiting NLRP3 inflammasome activation. NEK7 is a vital mediator of NLRP3 inflammasome activation, and glibenclamide or oridonin may be candidates for the development of new therapeutics against VILI driven by the interaction between NEK7 and NLRP3.


Assuntos
Anti-Inflamatórios/farmacologia , Diterpenos do Tipo Caurano/farmacologia , Glibureto/farmacologia , Quinases Relacionadas a NIMA/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Lesão Pulmonar Induzida por Ventilação Mecânica/tratamento farmacológico , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Animais , Fenômenos Biomecânicos , Cátions Monovalentes , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Transporte de Íons/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quinases Relacionadas a NIMA/antagonistas & inibidores , Quinases Relacionadas a NIMA/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Potássio/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Respiração Artificial/efeitos adversos , Transdução de Sinais , Lesão Pulmonar Induzida por Ventilação Mecânica/etiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia
19.
J Leukoc Biol ; 108(1): 283-295, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32237257

RESUMO

1,25-dihydroxyvitamin D3 (1,25(OH)2 D3, VitD3) is the major active ingredient of vitamin D and has anti-inflammatory activity; however, the mechanism for this remains poorly understood. In this study, we found that VitD3 was able to abolish NOD-like receptor protein 3 (NLRP3) inflammasome activation and subsequently inhibit caspase-1 activation and IL-1ß secretion via the vitamin D receptor (VDR). Furthermore, VitD3 specifically prevented NLRP3-mediated apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) oligomerization. In additional to this, NLRP3 binding to NIMA-related kinase 7 (NEK7) was also inhibited. Notably, VitD3 inhibited autophagy, leading to the inhibition of the NLRP3 inflammasome. Uncoupling protein 2-reactive oxygen species signaling may be involved in inflammasome suppression by VitD3. Importantly, VitD3 had both preventive and therapeutic effects on mouse model of ulcerative colitis, via inhibition of NLRP3 inflammasome activation. Our results reveal a mechanism through which VitD3 represses inflammation and prevents the relevant diseases, and suggest a potential clinical use of VitD3 in autoimmune syndromes or other NLRP3 inflammasome-driven inflammatory diseases.


Assuntos
Calcitriol/uso terapêutico , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Autofagia/efeitos dos fármacos , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Calcitriol/farmacologia , Caspase 1/metabolismo , Polaridade Celular/efeitos dos fármacos , Colite Ulcerativa/enzimologia , Colite Ulcerativa/imunologia , Colo/efeitos dos fármacos , Colo/patologia , Sulfato de Dextrana , Ativação Enzimática/efeitos dos fármacos , Interleucina-1beta/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/patologia , Camundongos , Quinases Relacionadas a NIMA/metabolismo , Proteólise/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Ubiquitinação/efeitos dos fármacos , Proteína Desacopladora 2/metabolismo
20.
Nature ; 580(7804): 542-547, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32322059

RESUMO

Prolonged mitosis often results in apoptosis1. Shortened mitosis causes tumorigenic aneuploidy, but it is unclear whether it also activates the apoptotic machinery2. Separase, a cysteine protease and trigger of all eukaryotic anaphases, has a caspase-like catalytic domain but has not previously been associated with cell death3,4. Here we show that human cells that enter mitosis with already active separase rapidly undergo death in mitosis owing to direct cleavage of anti-apoptotic MCL1 and BCL-XL by separase. Cleavage not only prevents MCL1 and BCL-XL from sequestering pro-apoptotic BAK, but also converts them into active promoters of death in mitosis. Our data strongly suggest that the deadliest cleavage fragment, the C-terminal half of MCL1, forms BAK/BAX-like pores in the mitochondrial outer membrane. MCL1 and BCL-XL are turned into separase substrates only upon phosphorylation by NEK2A. Early mitotic degradation of this kinase is therefore crucial for preventing apoptosis upon scheduled activation of separase in metaphase. Speeding up mitosis by abrogation of the spindle assembly checkpoint results in a temporal overlap of the enzymatic activities of NEK2A and separase and consequently in cell death. We propose that NEK2A and separase jointly check on spindle assembly checkpoint integrity and eliminate cells that are prone to chromosome missegregation owing to accelerated progression through early mitosis.


Assuntos
Apoptose , Mitose , Separase/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Segregação de Cromossomos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Fosforilação , Especificidade por Substrato , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/metabolismo
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