RESUMO
O-GlcNAc is a dynamic post-translational modification (PTM) that regulates protein functions. In studying the regulatory roles of O-GlcNAc, a major roadblock is the inability to change O-GlcNAcylation on a single protein at a time. Herein, we developed a dual RNA-aptamer-based approach that simultaneously targeted O-GlcNAc transferase (OGT) and ß-catenin, the key transcription factor of the Wnt signaling pathway, to selectively increase O-GlcNAcylation of the latter without affecting other OGT substrates. Using the OGT/ß-catenin dual-specificity aptamers, we found that O-GlcNAcylation of ß-catenin stabilizes the protein by inhibiting its interaction with ß-TrCP. O-GlcNAc also increases ß-catenin's interaction with EZH2, recruits EZH2 to promoters, and dramatically alters the transcriptome. Further, by coupling riboswitches or an inducible expression system to aptamers, we enabled inducible regulation of protein-specific O-GlcNAcylation. Together, our findings demonstrate the efficacy and versatility of dual-specificity aptamers for regulating O-GlcNAcylation on individual proteins.
Assuntos
Aptâmeros de Nucleotídeos , beta Catenina/metabolismo , Processamento de Proteína Pós-Traducional , Via de Sinalização Wnt , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/metabolismoRESUMO
The Wnt pathway is central to a host of developmental and disease-related processes. The remarkable conservation of this intercellular signaling cascade throughout metazoan lineages indicates that it coevolved with multicellularity to regulate the generation and spatial arrangement of distinct cell types. By regulating cell fate specification, mitotic activity, and cell polarity, Wnt signaling orchestrates development and tissue homeostasis, and its dysregulation is implicated in developmental defects, cancer, and degenerative disorders. We review advances in our understanding of this key pathway, from Wnt protein production and secretion to relay of the signal in the cytoplasm of the receiving cell. We discuss the evolutionary history of this pathway as well as endogenous and synthetic modulators of its activity. Finally, we highlight remaining gaps in our knowledge of Wnt signal transduction and avenues for future research.
Assuntos
Neoplasias , Via de Sinalização Wnt , Animais , Diferenciação Celular , Neoplasias/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismoRESUMO
This year's Gairdner Foundation Award for Biomedical Research goes to Roel Nusse for his pioneering work on the Wnt signaling pathway and its many roles in development, cancer, and stem cells.
Assuntos
Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Bibliografias como Assunto , Comunicação Celular , Drosophila , Proteínas de Drosophila/metabolismo , Feminino , Humanos , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Proteína Wnt1/metabolismoRESUMO
The T cell-specific DNA-binding protein TCF-1 is a central regulator of T cell development and function along multiple stages and lineages. Because it interacts with ß-catenin, TCF-1 has been classically viewed as a downstream effector of canonical Wnt signaling, although there is strong evidence for ß-catenin-independent TCF-1 functions. TCF-1 co-binds accessible regulatory regions containing or lacking its conserved motif and cooperates with other nuclear factors to establish context-dependent epigenetic and transcription programs that are essential for T cell development and for regulating immune responses to infection, autoimmunity and cancer. Although it has mostly been associated with positive regulation of chromatin accessibility and gene expression, TCF-1 has the potential to reduce chromatin accessibility and thereby suppress gene expression. In addition, the binding of TCF-1 bends the DNA and affects the chromatin conformation genome wide. This Review discusses the current understanding of the multiple roles of TCF-1 in T cell development and function and their mechanistic underpinnings.
Assuntos
Proteínas Wnt , beta Catenina , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
We performed the first proteogenomic characterization of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) using paired tumor and adjacent liver tissues from 159 patients. Integrated proteogenomic analyses revealed consistency and discordance among multi-omics, activation status of key signaling pathways, and liver-specific metabolic reprogramming in HBV-related HCC. Proteomic profiling identified three subgroups associated with clinical and molecular attributes including patient survival, tumor thrombus, genetic profile, and the liver-specific proteome. These proteomic subgroups have distinct features in metabolic reprogramming, microenvironment dysregulation, cell proliferation, and potential therapeutics. Two prognostic biomarkers, PYCR2 and ADH1A, related to proteomic subgrouping and involved in HCC metabolic reprogramming, were identified. CTNNB1 and TP53 mutation-associated signaling and metabolic profiles were revealed, among which mutated CTNNB1-associated ALDOA phosphorylation was validated to promote glycolysis and cell proliferation. Our study provides a valuable resource that significantly expands the knowledge of HBV-related HCC and may eventually benefit clinical practice.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Frutose-Bifosfato Aldolase/genética , Vírus da Hepatite B , Hepatite B Crônica/complicações , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Proteogenômica/métodos , beta Catenina/genética , Animais , Proliferação de Células , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Microambiente Tumoral/genéticaRESUMO
During corticogenesis, ventricular zone progenitors sequentially generate distinct subtypes of neurons, accounting for the diversity of neocortical cells and the circuits they form. While activity-dependent processes are critical for the differentiation and circuit assembly of postmitotic neurons, how bioelectrical processes affect nonexcitable cells, such as progenitors, remains largely unknown. Here, we reveal that, in the developing mouse neocortex, ventricular zone progenitors become more hyperpolarized as they generate successive subtypes of neurons. Experimental in vivo hyperpolarization shifted the transcriptional programs and division modes of these progenitors to a later developmental status, with precocious generation of intermediate progenitors and a forward shift in the laminar, molecular, morphological, and circuit features of their neuronal progeny. These effects occurred through inhibition of the Wnt-beta-catenin signaling pathway by hyperpolarization. Thus, during corticogenesis, bioelectric membrane properties are permissive for specific molecular pathways to coordinate the temporal progression of progenitor developmental programs and thus neocortical neuron diversity.
Assuntos
Potenciais da Membrana , Neocórtex/embriologia , Neurônios/metabolismo , Células-Tronco/citologia , Animais , Encéfalo/citologia , Encéfalo/embriologia , Diferenciação Celular , Progressão da Doença , Eletroporação , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Neocórtex/citologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Neurogênese , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Fatores de Tempo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Intestinal stem cells (ISCs) are maintained by stemness signaling for precise modulation of self-renewal and differentiation under homeostasis. However, the way in which intestinal immune cells regulate the self-renewal of ISCs remains elusive. Here we found that mouse and human Lgr5+ ISCs showed high expression of the immune cell-associated circular RNA circPan3 (originating from the Pan3 gene transcript). Deletion of circPan3 in Lgr5+ ISCs impaired their self-renewal capacity and the regeneration of gut epithelium in a manner dependent on immune cells. circPan3 bound mRNA encoding the cytokine IL-13 receptor subunit IL-13Rα1 (Il13ra1) in ISCs to increase its stability, which led to the expression of IL-13Rα1 in ISCs. IL-13 produced by group 2 innate lymphoid cells in the crypt niche engaged IL-13Rα1 on crypt ISCs and activated signaling mediated by IL-13âIL-13R, which in turn initiated expression of the transcription factor Foxp1. Foxp1 is associated with ß-catenin in rendering its nuclear translocation, which caused activation of the ß-catenin pathway and the maintenance of Lgr5+ ISCs.
Assuntos
Autorrenovação Celular/imunologia , Interleucina-13/metabolismo , Mucosa Intestinal/imunologia , RNA/metabolismo , Células-Tronco/fisiologia , Animais , Proteínas de Transporte/genética , Diferenciação Celular/imunologia , Autorrenovação Celular/genética , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-13/imunologia , Subunidade alfa1 de Receptor de Interleucina-13/genética , Subunidade alfa1 de Receptor de Interleucina-13/imunologia , Subunidade alfa1 de Receptor de Interleucina-13/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , RNA/genética , RNA/imunologia , RNA Circular , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regeneração/genética , Regeneração/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , beta Catenina/imunologia , beta Catenina/metabolismoRESUMO
Biomolecular condensates have emerged as a major organizational principle in the cell. However, the formation, maintenance, and dissolution of condensates are still poorly understood. Transcriptional machinery partitions into biomolecular condensates at key cell identity genes to activate these. Here, we report a specific perturbation of WNT-activated ß-catenin condensates that disrupts oncogenic signaling. We use a live-cell condensate imaging method in human cancer cells to discover FOXO and TCF-derived peptides that specifically inhibit ß-catenin condensate formation on DNA, perturb nuclear ß-catenin condensates in cells, and inhibit ß-catenin-driven transcriptional activation and colorectal cancer cell growth. We show that these peptides compete with homotypic intermolecular interactions that normally drive condensate formation. Using this framework, we derive short peptides that specifically perturb condensates and transcriptional activation of YAP and TAZ in the Hippo pathway. We propose a "monomer saturation" model in which short interacting peptides can be used to specifically inhibit condensate-associated transcription in disease.
Assuntos
Neoplasias , beta Catenina , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Transdução de Sinais , Via de Sinalização Hippo , Peptídeos/genéticaRESUMO
Zhu and Hart1 use dual-specificity RNA aptamers to recruit cellular O-GlcNAc transferase (OGT) and induce O-GlcNAc on target proteins like ß-catenin, revealing that O-GlcNAc stabilizes ß-catenin and enhances its transcriptional activity.
Assuntos
Aptâmeros de Nucleotídeos , N-Acetilglucosaminiltransferases , beta Catenina , N-Acetilglucosaminiltransferases/químicaRESUMO
Foxp3+ regulatory T cells (Treg cells) are the central component of peripheral immune tolerance. Whereas a dysregulated Treg cytokine signature has been observed in autoimmune diseases, the regulatory mechanisms underlying pro- and anti-inflammatory cytokine production are elusive. Here, we identify an imbalance between the cytokines IFN-γ and IL-10 as a shared Treg signature present in patients with multiple sclerosis and under high-salt conditions. RNA-sequencing analysis on human Treg subpopulations revealed ß-catenin as a key regulator of IFN-γ and IL-10 expression. The activated ß-catenin signature was enriched in human IFN-γ+ Treg cells, as confirmed in vivo with Treg-specific ß-catenin-stabilized mice exhibiting lethal autoimmunity with a dysfunctional Treg phenotype. Moreover, we identified prostaglandin E receptor 2 (PTGER2) as a regulator of IFN-γ and IL-10 production under a high-salt environment, with skewed activation of the ß-catenin-SGK1-Foxo axis. Our findings reveal a novel PTGER2-ß-catenin loop in Treg cells linking environmental high-salt conditions to autoimmunity.
Assuntos
Autoimunidade/imunologia , Inflamação/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Linfócitos T Reguladores/imunologia , beta Catenina/imunologia , Animais , Regulação da Expressão Gênica/imunologia , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-10/biossíntese , Interleucina-10/imunologia , Camundongos Endogâmicos C57BL , Receptores de Prostaglandina E Subtipo EP2/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
Understanding the contribution of the host's genetic background to cancer immunity may lead to improved stratification for immunotherapy and to the identification of novel therapeutic targets. We investigated the effect of common and rare germline variants on 139 well-defined immune traits in â¼9000 cancer patients enrolled in TCGA. High heritability was observed for estimates of NK cell and T cell subset infiltration and for interferon signaling. Common variants of IFIH1, TMEM173 (STING1), and TMEM108 were associated with differential interferon signaling and variants mapping to RBL1 correlated with T cell subset abundance. Pathogenic or likely pathogenic variants in BRCA1 and in genes involved in telomere stabilization and Wnt-ß-catenin also acted as immune modulators. Our findings provide evidence for the impact of germline genetics on the composition and functional orientation of the tumor immune microenvironment. The curated datasets, variants, and genes identified provide a resource toward further understanding of tumor-immune interactions.
Assuntos
Mutação em Linhagem Germinativa/genética , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica , Genes BRCA1 , Estudo de Associação Genômica Ampla , Humanos , Interferons/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Característica Quantitativa Herdável , Proteína p107 Retinoblastoma-Like/genética , Transdução de Sinais/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The transcriptional co-activators YAP and TAZ are key regulators of organ size and tissue homeostasis, and their dysregulation contributes to human cancer. Here, we discover YAP/TAZ as bona fide downstream effectors of the alternative Wnt signaling pathway. Wnt5a/b and Wnt3a induce YAP/TAZ activation independent of canonical Wnt/ß-catenin signaling. Mechanistically, we delineate the "alternative Wnt-YAP/TAZ signaling axis" that consists of Wnt-FZD/ROR-Gα12/13-Rho GTPases-Lats1/2 to promote YAP/TAZ activation and TEAD-mediated transcription. YAP/TAZ mediate the biological functions of alternative Wnt signaling, including gene expression, osteogenic differentiation, cell migration, and antagonism of Wnt/ß-catenin signaling. Together, our work establishes YAP/TAZ as critical mediators of alternative Wnt signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Via de Sinalização Wnt , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Receptores Frizzled/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Transativadores , Fatores de Transcrição , Proteínas de Sinalização YAP , beta Catenina/metabolismoRESUMO
Clinically acquired resistance to MAPK inhibitor (MAPKi) therapies for melanoma cannot be fully explained by genomic mechanisms and may be accompanied by co-evolution of intra-tumoral immunity. We sought to discover non-genomic mechanisms of acquired resistance and dynamic immune compositions by a comparative, transcriptomic-methylomic analysis of patient-matched melanoma tumors biopsied before therapy and during disease progression. Transcriptomic alterations across resistant tumors were highly recurrent, in contrast to mutations, and were frequently correlated with differential methylation of tumor cell-intrinsic CpG sites. We identified in the tumor cell compartment supra-physiologic c-MET up-expression, infra-physiologic LEF1 down-expression and YAP1 signature enrichment as drivers of acquired resistance. Importantly, high intra-tumoral cytolytic T cell inflammation prior to MAPKi therapy preceded CD8 T cell deficiency/exhaustion and loss of antigen presentation in half of disease-progressive melanomas, suggesting cross-resistance to salvage anti-PD-1/PD-L1 immunotherapy. Thus, melanoma acquires MAPKi resistance with highly dynamic and recurrent non-genomic alterations and co-evolving intra-tumoral immunity.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Linfócitos T CD8-Positivos/imunologia , Metilação de DNA , Perfilação da Expressão Gênica , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Melanoma/imunologia , Fosfoproteínas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Transcrição , Proteínas de Sinalização YAP , beta Catenina/metabolismoRESUMO
Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.
Assuntos
Carcinoma Hepatocelular , Progressão da Doença , Elasticidade , Matriz Extracelular , Cirrose Hepática , Neoplasias Hepáticas , Animais , Humanos , beta Catenina/metabolismo , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Colágeno/química , Colágeno/metabolismo , Simulação por Computador , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Matriz Extracelular/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Integrina beta1/metabolismo , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Invasividade Neoplásica , Viscosidade , Proteínas de Sinalização YAP/metabolismo , Cirrose Hepática/complicações , Cirrose Hepática/metabolismo , Cirrose Hepática/patologiaRESUMO
HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of ß-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced ß-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Leucemia Mieloide Aguda/metabolismo , Estruturas R-Loop , RNA Longo não Codificante/metabolismo , beta Catenina/metabolismo , Animais , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos Transgênicos , RNA Longo não Codificante/genética , Relação Estrutura-Atividade , Transcrição Gênica , Ativação Transcricional , beta Catenina/genética , CoesinasRESUMO
The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the ß-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for ß-TrCP recruitment to the complex and ß-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/ß-catenin signaling. In line, the ß-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Aciltransferases , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Proteínas de Sinalização YAPRESUMO
The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating ß-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Carboidratos da Dieta/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Butiratos/metabolismo , Proliferação de Células , Transformação Celular Neoplásica , Pólipos do Colo/metabolismo , Pólipos do Colo/microbiologia , Pólipos do Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Reparo de Erro de Pareamento de DNA , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/metabolismo , Organismos Livres de Patógenos Específicos , beta Catenina/metabolismoRESUMO
The Wnt/ß-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by ß-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein ß-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human ß-catenin destruction complex from purified components, recapitulating complex assembly, ß-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote ß-catenin capture, phosphorylation, and ubiquitylation. APC facilitates ß-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFß-TrCP E3 ligase complex in a ß-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes ß-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/ß-catenin signaling.