RESUMO
The activation of the NLRP3 inflammasome plays a crucial role in the innate immune response. During cell division, NLRP3 inflammasome activation must be strictly controlled. In this study, we discover that the anaphase-promoting complex subunit 10 (APC10), a substrate recognition protein of the anaphase-promoting complex/cyclosome (APC/C), is a critical mediator of NLRP3 inflammasome activation. During interphase, APC10 interacts with NLRP3 to promote NLRP3 inflammasome activation, whereas during mitosis, APC10 disassociates from the NLRP3 inflammasome to repress inflammatory responses. This study reveals a distinct mechanism by which APC10 serves as a switch for NLRP3 inflammasome activation during the cell cycle.
Assuntos
Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclo Celular , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Humanos , UbiquitinaçãoRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif-dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.
Assuntos
Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Ciclo Celular , Proteínas de Ciclo Celular/química , Mapas de Interação de Proteínas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Especificidade por Substrato , UbiquitinaçãoRESUMO
The anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase, is responsible for the transition from metaphase to anaphase and the exit from mitosis. The anaphase promoting complex subunit 10 (APC10), a subunit of the APC/C, executes a vital function in substrate recognition. However, no research has reported the connection between APC10 and cancer until now. In this study, we uncovered a novel, unprecedented role of APC10 in tumor progression, which is independent of APC/C. First, aberrant increase of APC10 expression was validated in non-small cell lung cancer (NSCLC) cells and tissues, and the absence of APC10 repressed cell proliferation and migration. Of great interest, we found that APC10 inhibition induced cell cycle arrest at the G0/G1 phase and reduced the expression of the APC/C substrate, Cyclin B1; this finding is different from the conventional concept of the accumulation of Cyclin B1 and cell cycle arrest in metaphase. Further, APC10 was found to interact with glutaminase C (GAC), and the inhibition of APC10 weakened glutamine metabolism and induced excessive autophagy. Taken together, these findings identify a novel function of APC10 in the regulation of NSCLC tumorigenesis and point to the possibility of APC10 as a new target for cancer therapy.
Assuntos
Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Carcinogênese/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Progressão da Doença , Neoplasias Pulmonares/metabolismo , Células A549 , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/genética , Autofagia/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/genética , Proliferação de Células/genética , Citoplasma/metabolismo , Fase G1/genética , Glutaminase/metabolismo , Glutamina/metabolismo , Humanos , Neoplasias Pulmonares/patologia , RNA Interferente Pequeno/genética , Fase de Repouso do Ciclo Celular/genética , Transdução de Sinais/genética , TransfecçãoRESUMO
Sox2 has a critical role in embryonic stem (ES) cell maintenance and differentiation. Interestingly, its activity is highly dosage-dependent. Although transcriptional regulation of Sox2 has been extensively studied, the mechanisms orchestrating its degradation remain unclear. In this study, we identified ubiquitin-conjugating enzyme E2S (Ube2s) as a novel effector for Sox2 protein degradation. Ube2s mediates K11-linked polyubiquitin chain formation at the Sox2-K123 residue, thus marking it for proteasome-mediated degradation. Besides its role in fine-tuning the precise level of Sox2, Ube2s reinforces the self-renewing and pluripotent state of ES cells. Importantly, it also represses Sox2-mediated ES cell differentiation toward the neural ectodermal lineage.
Assuntos
Células-Tronco Embrionárias Murinas/fisiologia , Fatores de Transcrição SOXB1/metabolismo , Enzimas de Conjugação de Ubiquitina/fisiologia , Animais , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Camundongos , Estabilidade Proteica , Proteólise , UbiquitinaçãoRESUMO
The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex and interphase early mitotic inhibitor 1 (Emi1) ensures the correct order and timing of distinct cell-cycle transitions. Here we use cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of future experiments to investigate APC/C functions in vivo.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Ubiquitinação , Ciclossomo-Complexo Promotor de Anáfase/química , Antígenos CD , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Caderinas/química , Caderinas/metabolismo , Caderinas/ultraestrutura , Domínio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Proteínas F-Box/ultraestrutura , Humanos , Lisina/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina/ultraestrutura , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/ultraestruturaRESUMO
The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of the G1 phase of the cell cycle. The APC/C is an unusually large multimeric cullin-RING ligase. Its activity is strictly dependent on regulatory coactivator subunits that promote APC/C-substrate interactions and stimulate its catalytic reaction. Because the structures of many APC/C subunits and their organization within the assembly are unknown, the molecular basis for these processes is poorly understood. Here, from a cryo-electron microscopy reconstruction of a human APC/C-coactivator-substrate complex at 7.4 Å resolution, we have determined the complete secondary structural architecture of the complex. With this information we identified protein folds for structurally uncharacterized subunits, and the definitive location of all 20 APC/C subunits within the 1.2 MDa assembly. Comparison with apo APC/C shows that the coactivator promotes a profound allosteric transition involving displacement of the cullin-RING catalytic subunits relative to the degron-recognition module of coactivator and APC10. This transition is accompanied by increased flexibility of the cullin-RING subunits and enhanced affinity for UBCH10-ubiquitin, changes which may contribute to coactivator-mediated stimulation of APC/C E3 ligase activity.