RESUMO
For many years, a connection between circadian clocks and cancer has been postulated. Here we describe an unexpected function for the circadian repressor CRY2 as a component of an FBXL3-containing E3 ligase that recruits T58-phosphorylated c-MYC for ubiquitylation. c-MYC is a critical regulator of cell proliferation; T58 is central in a phosphodegron long recognized as a hotspot for mutation in cancer. This site is also targeted by FBXW7, although the full machinery responsible for its turnover has remained obscure. CRY1 cannot substitute for CRY2 in promoting c-MYC degradation. Their unique functions may explain prior conflicting reports that have fueled uncertainty about the relationship between clocks and cancer. We demonstrate that c-MYC is a target of CRY2-dependent protein turnover, suggesting a molecular mechanism for circadian control of cell growth and a new paradigm for circadian protein degradation.
Assuntos
Transformação Celular Neoplásica/genética , Relógios Circadianos/genética , Criptocromos/genética , Proteínas F-Box/genética , Regulação Neoplásica da Expressão Gênica , Linfoma/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Ritmo Circadiano/genética , Criptocromos/química , Criptocromos/metabolismo , Proteínas Culina/química , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Fibroblastos , Células HEK293 , Humanos , Linfoma/metabolismo , Linfoma/mortalidade , Linfoma/patologia , Camundongos , Camundongos Knockout , Modelos Moleculares , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteólise , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Quinases Associadas a Fase S/química , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Análise de SobrevidaRESUMO
As solid tumors expand, oxygen and nutrients become limiting owing to inadequate vascularization and diffusion. How malignant cells cope with this potentially lethal metabolic stress remains poorly understood. We found that glucose shortage associated with malignant progression triggers apoptosis through the endoplasmic reticulum (ER) unfolded protein response (UPR). ER stress is in part caused by reduced glucose flux through the hexosamine pathway. Deletion of the proapoptotic UPR effector CHOP in a mouse model of K-ras(G12V)-induced lung cancer increases tumor incidence, strongly supporting the notion that ER stress serves as a barrier to malignancy. Overcoming this barrier requires the selective attenuation of the PERK-CHOP arm of the UPR by the molecular chaperone p58(IPK). Furthermore, p58(IPK)-mediated adaptive response enables cells to benefit from the protective features of chronic UPR. Altogether, these results show that ER stress activation and p58(IPK) expression control the fate of malignant cells facing glucose shortage.
Assuntos
Apoptose , Transformação Celular Neoplásica/metabolismo , Glucose/deficiência , Chaperonas Moleculares/fisiologia , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo , Acetilgalactosamina/metabolismo , Animais , Hipóxia Celular , Linhagem Celular , Proliferação de Células , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Transportador de Glucose Tipo 1/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Ácido Láctico/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-ret/metabolismo , Ratos , Resposta a Proteínas não DobradasRESUMO
Nuclear hormone receptors (NRs) regulate physiology by sensing lipophilic ligands and adapting cellular transcription appropriately. A growing understanding of the impact of circadian clocks on mammalian transcription has sparked interest in the interregulation of transcriptional programs. Mammalian clocks are based on a transcriptional feedback loop featuring the transcriptional activators circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), and transcriptional repressors cryptochrome (CRY) and period (PER). CRY1 and CRY2 bind independently of other core clock factors to many genomic sites, which are enriched for NR recognition motifs. Here we report that CRY1/2 serve as corepressors for many NRs, indicating a new facet of circadian control of NR-mediated regulation of metabolism and physiology, and specifically contribute to diurnal modulation of drug metabolism.
Assuntos
Proteínas CLOCK/metabolismo , Ritmo Circadiano/fisiologia , Criptocromos/metabolismo , Proteínas Circadianas Period/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica/fisiologia , Fatores de Transcrição ARNTL/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Relógios Circadianos/fisiologia , Retroalimentação Fisiológica/fisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Transativadores/metabolismoRESUMO
Eukaryotic cells encounter diverse threats jeopardizing their integrity, prompting the development of defense mechanisms against these stressors. Among these mechanisms, inflammasomes are well-known for their roles in coordinating the inflammatory response against infections. Extensive research has unveiled their multifaceted involvement in cellular processes beyond inflammation. Recent studies emphasize the intricate relationship between the inflammasome and the DNA damage response (DDR). They highlight how the DDR participates in inflammasome activation and the reciprocal impact of inflammasome on DDR and genome integrity preservation. Moreover, novel functions of inflammasome sensors in DDR pathways have emerged, broadening our understanding of their roles. Finally, this review delves into identifying common signals that drive the activation of inflammasome sensors alongside activation cues for the DNA damage response, offering potential insights into shared regulatory pathways between these critical cellular processes.
RESUMO
Inflammasomes are multiprotein platforms that control caspase-1 activation, which process the inactive precursor forms of the inflammatory cytokines IL-1ß and IL-18, leading to an inflammatory type of programmed cell death called pyroptosis. Studying inflammasome-driven processes, such as pyroptosis-induced cell swelling, under controlled conditions remains challenging because the signals that activate pyroptosis also stimulate other signaling pathways. We designed an optogenetic approach using a photo-oligomerizable inflammasome core adapter protein, apoptosis-associated speck-like containing a caspase recruitment domain (ASC), to temporally and quantitatively manipulate inflammasome activation. We demonstrated that inducing the light-sensitive oligomerization of ASC was sufficient to recapitulate the classical features of inflammasomes within minutes. This system showed that there were two phases of cell swelling during pyroptosis. This approach offers avenues for biophysical investigations into the intricate nature of cellular volume control and plasma membrane rupture during cell death.
Assuntos
Proteínas Adaptadoras de Sinalização CARD , Inflamassomos , Optogenética , Piroptose , Inflamassomos/metabolismo , Optogenética/métodos , Animais , Humanos , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Camundongos , Caspase 1/metabolismo , Caspase 1/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/genéticaRESUMO
NLRP3 is a pattern recognition receptor with a well-documented role in inducing inflammasome assembly in response to cellular stress. Deregulation of its activity leads to many inflammatory disorders including gouty arthritis, Alzheimer disease, and cancer. Whereas its role in the context of cancer has been mostly explored in the immune compartment, whether NLRP3 exerts functions unrelated to immunity in cancer development remains unexplored. Here, we demonstrate that NLRP3 interacts with the ATM kinase to control the activation of the DNA damage response, independently of its inflammasome activity. NLRP3 down-regulation in both broncho- and mammary human epithelial cells significantly impairs ATM pathway activation, leading to lower p53 activation, and provides cells with the ability to resist apoptosis induced by acute genotoxic stress. Interestingly, NLRP3 expression is down-regulated in non-small cell lung cancers and breast cancers, and its expression positively correlates with patient overall survival. Our findings identify a novel non-immune function for NLRP3 in maintaining genome integrity and strengthen the concept of a functional link between innate immunity and DNA damage sensing pathways to maintain cell integrity.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Imunidade Inata , Dano ao DNA , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Early 2 factor (E2F) family transcription factors participate in myriad cell biological processes including: the cell cycle, DNA repair, apoptosis, development, differentiation, and metabolism. Circadian rhythms influence many of these phenomena. Here we find that a mammalian circadian rhythm component, Cryptochrome 2 (CRY2), regulates E2F family members. Furthermore, CRY1 and CRY2 cooperate with the E3 ligase complex SKP-CULLIN-FBXL3 (SCFFBXL3) to reduce E2F steady state protein levels. These findings reveal an unrecognized molecular connection between circadian clocks and cell cycle regulation and highlight another mechanism to maintain appropriate E2F protein levels for proper cell growth.
Assuntos
Ritmo Circadiano , Criptocromos/fisiologia , Fatores de Transcrição E2F/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Animais , Fatores de Transcrição E2F/genética , Camundongos Knockout , Fatores de Transcrição/genética , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
We recently demonstrated that the circadian clock component CRY2 is an essential cofactor in the SCFFBXL3-mediated ubiquitination of c-MYC. Because our demonstration that CRY2 recruits phosphorylated substrates to SCFFBXL3 was unexpected, we investigated the scope of this role by searching for additional substrates of FBXL3 that require CRY1 or CRY2 as cofactors. Here, we describe an affinity purification mass spectrometry (APMS) screen through which we identified more than one hundred potential substrates of SCFFBXL3+CRY1/2, including the cell cycle regulated Tousled-like kinase, TLK2. Both CRY1 and CRY2 recruit TLK2 to SCFFBXL3, and TLK2 kinase activity is required for this interaction. Overexpression or genetic deletion of CRY1 and/or CRY2 decreases or enhances TLK2 protein abundance, respectively. These findings reinforce the idea that CRYs function as co-factors for SCFFBXL3, provide a resource of potential substrates, and establish a molecular connection between the circadian and cell cycle oscillators via CRY-modulated turnover of TLK2.
Assuntos
Criptocromos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Animais , Células Cultivadas , Relógios Circadianos , Criptocromos/genética , Proteínas F-Box/metabolismo , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Fator de Células-Tronco/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Cellular metabolite balance and mitochondrial function are under circadian control, but the pathways connecting the molecular clock to these functions are unclear. Peroxisome proliferator-activated receptor delta (PPARδ) enables preferential utilization of lipids as fuel during exercise and is a major driver of exercise endurance. We show here that the circadian repressors CRY1 and CRY2 function as co-repressors for PPARδ. Cry1-/-;Cry2-/- myotubes and muscles exhibit elevated expression of PPARδ target genes, particularly in the context of exercise. Notably, CRY1/2 seem to repress a distinct subset of PPARδ target genes in muscle compared to the co-repressor NCOR1. In vivo, genetic disruption of Cry1 and Cry2 enhances sprint exercise performance in mice. Collectively, our data demonstrate that CRY1 and CRY2 modulate exercise physiology by altering the activity of several transcription factors, including CLOCK/BMAL1 and PPARδ, and thereby alter energy storage and substrate selection for energy production.
Assuntos
Criptocromos/metabolismo , PPAR delta/metabolismo , Condicionamento Físico Animal , Animais , Células Cultivadas , Criptocromos/genética , Deleção de Genes , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculos/fisiologia , Mapas de Interação de ProteínasRESUMO
Metformin is widely used in the treatment of type 2 diabetes to lower blood glucose. Although metformin is a relatively safe and effective drug, its clinical efficacy is variable and under certain circumstances it may contribute to life-threatening lactic acidosis. Thus, additional understanding of metformin pharmacokinetics and pharmacodynamics could provide important information regarding therapeutic use of this widely prescribed drug. Here we report a significant effect of time of day on acute blood glucose reduction in response to metformin administration and on blood lactate levels in healthy mice. Furthermore, we demonstrate that while metformin transport into hepatocytes is unaltered by time of day, the kinetics of metformin-induced activation of AMP-activated protein kinase (AMPK) in the liver are remarkably altered with circadian time. Liver-specific ablation of Bmal1 expression alters metformin induction of AMPK and blood glucose response but does not completely abolish time of day differences. Together, these data demonstrate that circadian rhythms affect the biological responses to metformin in a complex manner.
Assuntos
Relógios Circadianos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/fisiologia , Metformina/administração & dosagem , Proteínas Quinases Ativadas por AMP , Animais , Glicemia/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Lactatos/sangue , Masculino , Camundongos , Proteínas Serina-Treonina QuinasesRESUMO
The circadian transcriptional repressors cryptochrome 1 (Cry1) and 2 (Cry2) evolved from photolyases, bacterial light-activated DNA repair enzymes. In this study, we report that while they have lost DNA repair activity, Cry1/2 adapted to protect genomic integrity by responding to DNA damage through posttranslational modification and coordinating the downstream transcriptional response. We demonstrate that genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by Herpes virus associated ubiquitin-specific protease (Hausp, a.k.a Usp7), stabilizing Cry1 and shifting circadian clock time. DNA damage also increases Cry2 interaction with Fbxl3, destabilizing Cry2. Thus, genotoxic stress increases the Cry1/Cry2 ratio, suggesting distinct functions for Cry1 and Cry2 following DNA damage. Indeed, the transcriptional response to genotoxic stress is enhanced in Cry1-/- and blunted in Cry2-/- cells. Furthermore, Cry2-/- cells accumulate damaged DNA. These results suggest that Cry1 and Cry2, which evolved from DNA repair enzymes, protect genomic integrity via coordinated transcriptional regulation.
Assuntos
Relógios Circadianos/genética , Criptocromos/fisiologia , Dano ao DNA , Proteases Específicas de Ubiquitina/fisiologia , Animais , Linhagem Celular , Criptocromos/metabolismo , Camundongos , Fosforilação , Ligação Proteica , Estabilidade Proteica , Transcrição Gênica , Peptidase 7 Específica de Ubiquitina , Proteases Específicas de Ubiquitina/metabolismoRESUMO
PURPOSE: Multiple myeloma is a clonal plasma cell disorder in which growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type I (IGF-I). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma. EXPERIMENTAL DESIGN: We analyzed the impact of IGF-I combined with proteasome inhibitors on multiple myeloma cell lines in vivo and in vitro as well as on fresh human myeloma cells. RESULTS: Our study shows that IGF-I enhances the cytotoxic effect of proteasome inhibitors against myeloma cells. The effect of bortezomib on the content of proapoptotic proteins such as Bax, Bad, Bak, and BimS and antiapoptotic proteins such as Bcl-2, Bcl-XL, XIAP, Bfl-1, and survivin was enhanced by IGF-I. The addition of IGF-I to bortezomib had a minor effect on NF-κB signaling in MM.1S cells while strongly enhancing reticulum stress. This resulted in an unfolded protein response (UPR), which was required for the potentiating effect of IGF-I on bortezomib cytotoxicity as shown by siRNA-mediated inhibition of GADD153 expression. CONCLUSIONS: These results suggest that the high baseline level of protein synthesis in myeloma can be exploited therapeutically by combining proteasome inhibitors with IGF-I, which possesses a "priming" effect on myeloma cells for this family of compounds.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Mieloma Múltiplo/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Bortezomib , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Humanos , Fator de Crescimento Insulin-Like I/administração & dosagem , Fator de Crescimento Insulin-Like I/toxicidade , Mieloma Múltiplo/tratamento farmacológico , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/toxicidade , Biossíntese de Proteínas/efeitos dos fármacos , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Cellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes-associated protein whose expression level affects cellular free cholesterol content. METHODOLOGY/PRINCIPAL FINDINGS: Using a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY. CONCLUSIONS/SIGNIFICANCE: LDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis.