RESUMEN
In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5' m(7)GTP cap-protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m(7)GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m(7)GTP-5(')cap and Ago1/2 within the miRISC complex attached to the 3'-UTR of mRNA, creating an inhibitory closed-loop complex.
Asunto(s)
Proteínas Portadoras/metabolismo , Silenciador del Gen , MicroARNs/metabolismo , Animales , Proteínas Portadoras/genética , Citoplasma/genética , Citoplasma/metabolismo , Genes , Péptidos y Proteínas de Señalización Intracelular , Proteínas con Dominio LIM , Ratones , MicroARNs/genética , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Loss of heterozygosity (LOH) and homozygous deletions at chromosome 3p21.3 are common in both small and nonsmall cell lung cancers, indicating the likely presence of tumor suppressor genes (TSGs). Although genetic and epigenetic changes within this region have been identified, the functional significance of these changes has not been explored. Concurrent protein expression and genetic analyses of human lung tumors coupled with functional studies have not been done. Here, we show that expression of the 3p21.3 gene, LIMD1, is frequently down-regulated in human lung tumors. Loss of LIMD1 expression occurs through a combination of gene deletion, LOH, and epigenetic silencing of transcription without evidence for coding region mutations. Experimentally, LIMD1 is a bona fide TSG. Limd1(-/-) mice are predisposed to chemical-induced lung adenocarcinoma and genetic inactivation of Limd1 in mice heterozygous for oncogenic K-Ras(G12D) markedly increased tumor initiation, promotion, and mortality. Thus, we conclude that LIMD1 is a validated chromosome 3p21.3 tumor-suppressor gene involved in human lung cancer development. LIMD1 is a LIM domain containing adapter protein that localizes to E-cadherin cell-cell adhesive junctions, yet also translocates to the nucleus where it has been shown to function as an RB corepressor. As such, LIMD1 has the potential to communicate cell extrinsic or environmental cues with nuclear responses.
Asunto(s)
Adenocarcinoma/genética , Cromosomas Humanos Par 3/genética , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/inducido químicamente , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Metilación de ADN , Eliminación de Gen , Silenciador del Gen , Genes ras , Predisposición Genética a la Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas con Dominio LIM , Pérdida de Heterocigocidad , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Ratones , Ratones Mutantes , Regiones Promotoras Genéticas , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patologíaRESUMEN
LIMD1 (LIM domain-containing protein 1) is considered as a tumor suppressor, being deregulated in many cancers to include hematological malignancies; however, very little is known about the underlying mechanisms of its deregulation and its roles in carcinogenesis. Epstein-Barr Virus (EBV) is associated with a panel of malignancies of lymphocytic and epithelial origin. Using high throughput expression profiling, we have previously identified LIMD1 as a common marker associated with the oncogenic transcription factor IRF4 in EBV-related lymphomas and other hematological malignancies. In this study, we have identified potential conserved IRF4- and NFκB-binding motifs in the LIMD1 gene promoter, and both are demonstrated functional by promoter-reporter assays. We further show that LIMD1 is partially upregulated by EBV latent membrane protein 1 (LMP1) via IRF4 and NFκB in EBV latency. As to its role in the setting of EBV latent infection, we show that LIMD1 interacts with TRAF6, a crucial mediator of LMP1 signal transduction. Importantly, LIMD1 depletion impairs LMP1 signaling and functions, potentiates ionomycin-induced DNA damage and apoptosis, and inhibits p62-mediated selective autophagy. Taken together, these results show that LIMD1 is upregulated in EBV latency and plays an oncogenic role rather than that of a tumor suppressor. Our findings have identified LIMD1 as a novel player in EBV latency and oncogenesis, and open a novel research avenue, in which LIMD1 and p62 play crucial roles in linking DNA damage response (DDR), apoptosis, and autophagy and their potential interplay during viral oncogenesis.
RESUMEN
The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-ß subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1-negative lung cancers.
Asunto(s)
Adenocarcinoma/genética , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , Neoplasias Pulmonares/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Retroalimentación Fisiológica , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Masculino , Ratones , Persona de Mediana Edad , Pronóstico , Análisis de Supervivencia , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
As core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.
Asunto(s)
Proteínas Argonautas/metabolismo , Silenciador del Gen , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , MicroARNs/genética , Proteínas Argonautas/genética , Autoantígenos/metabolismo , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/química , Proteínas con Dominio LIM/genética , MicroARNs/metabolismo , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O(2) tension. In high O(2) tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1α, which leads to binding of the von Hippel-Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin-ligase complex, initiating HIF-1α ubiquitylation and degradation. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1α or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD-LIMD1-VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1α. Depletion of endogenous LIMD1 increases HIF-1α levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxic regulators.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/genética , Modelos Biológicos , Poliubiquitina/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Interferencia de ARN , Transfección , Técnicas del Sistema de Dos Híbridos , Ubiquitinación , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
LIMD1 is a tumour suppressor gene (TSG) down regulated in â¼80% of lung cancers with loss also demonstrated in breast and head and neck squamous cell carcinomas. LIMD1 is also a candidate TSG in childhood acute lymphoblastic leukaemia. Mechanistically, LIMD1 interacts with pRB, repressing E2F-driven transcription as well as being a critical component of microRNA-mediated gene silencing. In this study we show a CpG island within the LIMD1 promoter contains a conserved binding motif for the transcription factor PU.1. Mutation of the PU.1 consensus reduced promoter driven transcription by 90%. ChIP and EMSA analysis demonstrated that PU.1 specifically binds to the LIMD1 promoter. siRNA depletion of PU.1 significantly reduced endogenous LIMD1 expression, demonstrating that PU.1 is a major transcriptional activator of LIMD1.