RESUMEN
ERI1 is a 3'-to-5' exoribonuclease involved in RNA metabolic pathways including 5.8S rRNA processing and turnover of histone mRNAs. Its biological and medical significance remain unclear. Here, we uncover a phenotypic dichotomy associated with bi-allelic ERI1 variants by reporting eight affected individuals from seven unrelated families. A severe spondyloepimetaphyseal dysplasia (SEMD) was identified in five affected individuals with missense variants but not in those with bi-allelic null variants, who showed mild intellectual disability and digital anomalies. The ERI1 missense variants cause a loss of the exoribonuclease activity, leading to defective trimming of the 5.8S rRNA 3' end and a decreased degradation of replication-dependent histone mRNAs. Affected-individual-derived induced pluripotent stem cells (iPSCs) showed impaired in vitro chondrogenesis with downregulation of genes regulating skeletal patterning. Our study establishes an entity previously unreported in OMIM and provides a model showing a more severe effect of missense alleles than null alleles within recessive genotypes, suggesting a key role of ERI1-mediated RNA metabolism in human skeletal patterning and chondrogenesis.
Asunto(s)
Exorribonucleasas , Histonas , Humanos , Exorribonucleasas/genética , Histonas/genética , Mutación Missense/genética , ARN Ribosómico 5.8S , ARN , ARN Mensajero/genéticaRESUMEN
Bioconjugates are an important class of therapeutic molecules. To date, O-glycan-based metabolic glycoengineering has had limited use in this field, due to the complexities of the endogenous O-glycosylation pathway and the lack of an O-glycosylation consensus sequence. Here, we describe the development of a versatile on-demand O-glycosylation system that uses a novel, widely applicable 5 amino acid O-glycosylation tag, and a metabolically engineered UDP-galactose-4-eperimase (GALE) knock-out cell line. Optimization of the primary sequence of the tag enables the production of Fc-based proteins with either single or multiple O-glycans with complexity fully controlled by media supplementation. We demonstrate how the uniformly labeled proteins containing exclusively N-azido-acetylgalactosamine are used for CLICK chemistry-based bioconjugation to generate site-specifically fluorochrome-labeled antibodies, dual-payload molecules, and bioactive Fc-peptides for applications in basic research and drug discovery. To our knowledge, this is the first description of generating a site-specific O-glycosylation system by combining an O-glycosylation tag and a metabolically engineered cell line.
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Química Clic , Polisacáridos , Glicosilación , Polisacáridos/químicaRESUMEN
A recombinant Newcastle Disease Virus (NDV), encoding either a human (NDVhuGM-CSF, MEDI5395) or murine (NDVmuGM-CSF) GM-CSF transgene, combined broad oncolytic activity with the ability to significantly modulate genes related to immune functionality in human tumor cells. Replication in murine tumor lines was significantly diminished relative to human tumor cells. Nonetheless, intratumoral injection of NDVmuGM-CSF conferred antitumor effects in three syngeneic models in vivo; with efficacy further augmented by concomitant treatment with anti-PD-1/PD-L1 or T-cell agonists. Ex vivo immune profiling, including T-cell receptor sequencing, revealed profound immune-contexture changes consistent with priming and potentiation of adaptive immunity and tumor microenvironment (TME) reprogramming toward an immune-permissive state. CRISPR modifications rendered CT26 tumors significantly more permissive to NDV replication, and in this setting, NDVmuGM-CSF confers immune-mediated effects in the noninjected tumor in vivo Taken together, the data support the thesis that MEDI5395 primes and augments cell-mediated antitumor immunity and has significant utility as a combination partner with other immunomodulatory cancer treatments.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Inmunomodulación , Inmunoterapia/métodos , Virus de la Enfermedad de Newcastle/genética , Viroterapia Oncolítica/instrumentación , Microambiente Tumoral , Animales , Apoptosis , Proliferación Celular , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage-associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour-suppressive mechanism.
Asunto(s)
Apoptosis , Carcinoma Hepatocelular/prevención & control , Forma de la Célula , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Neoplasias Hepáticas/prevención & control , Hígado/patología , Quinasas Asociadas a rho/metabolismo , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Caspasas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Dietilnitrosamina , Modelos Animales de Enfermedad , Activación Enzimática , Ácido Glicirrínico , Células HEK293 , Proteína HMGB1/metabolismo , Humanos , Hígado/enzimología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Mutación , Cadenas Ligeras de Miosina/metabolismo , Infiltración Neutrófila , Fosforilación , Sulfonamidas , Receptor Toll-Like 4/metabolismo , Quinasas Asociadas a rho/genéticaRESUMEN
ROCK2 roles in epidermal differentiation and carcinogenesis have been investigated in mice expressing an RU486-inducible, 4HT-activated ROCK2 transgene (K14.creP/lslROCKer). RU486/4HT-mediated ROCKer activation induced epidermal hyperplasia similar to cutaneous oncogenic rasHa (HK1.ras); however ROCKer did not elicit papillomas. Instead, anomalous basal-layer ROCKer expression corrupted normal ROCK2 roles underlying epidermal rigidity/stiffness and barrier maintanance, resulting in premature keratin K1, loricrin and filaggrin expression. Also, hyperproliferative/stress-associated keratin K6 was reduced; possibly reflecting altered ROCK2 roles in epidermal rigidity and keratinocyte flexibility/migration during wound healing. Consistent with increased proliferation, K14.creP/lslROCKer hyperplasia displayed supra-basal-to-basal increases in activated p-AKT1, inactivated p-GSK3ßâser9 and membranous/nuclear ß-catenin expression together with weak NFκB, which were absent in equivalent HK1.ras hyperplasia. Furthermore, ROCKer-mediated increases in epidermal rigidity via p-MypT1 inactivation/elevated MLC, coupled to anomalous ß-catenin expression, induced tenascin C-positive dermal fibroblasts. Alongside an altered ECM, these latent tenascin C-positive dermal fibroblasts may become putative pre-cancer-associated fibroblasts (pre-CAFs) and establish a susceptibility that subsequently contributes to tumour progression. However, anomalous differentiation was also accompanied by an immediate increase in basal-layer p53/p21 expression; suggesting that while ROCK2/AKT1/ß-catenin activation increased keratinocyte proliferation resulting in hyperplasia, compensatory p53/p21 and accelerated differentiation helped inhibit papillomatogenesis.
Asunto(s)
Carcinogénesis/metabolismo , Papiloma/metabolismo , Papiloma/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Quinasas Asociadas a rho/metabolismo , Animales , Carcinogénesis/patología , Diferenciación Celular , Epidermis/metabolismo , Epidermis/patología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tenascina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , beta Catenina/metabolismo , Quinasas Asociadas a rho/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease; the identification of novel targets and development of effective treatment strategies are urgently needed to improve patient outcomes. Remodeling of the pancreatic stroma occurs during PDAC development, which drives disease progression and impairs responses to therapy. The actomyosin regulatory ROCK1 and ROCK2 kinases govern cell motility and contractility, and have been suggested to be potential targets for cancer therapy, particularly to reduce the metastatic spread of tumor cells. However, ROCK inhibitors are not currently used for cancer patient treatment, largely due to the overwhelming challenge faced in the development of anti-metastatic drugs, and a lack of clarity as to the cancer types most likely to benefit from ROCK inhibitor therapy. In 2 recent publications, we discovered that ROCK1 and ROCK2 expression were increased in PDAC, and that increased ROCK activity was associated with reduced survival and PDAC progression by enabling extracellular matrix (ECM) remodeling and invasive growth of pancreatic cancer cells. We also used intravital imaging to optimize ROCK inhibition using the pharmacological ROCK inhibitor fasudil (HA-1077), and demonstrated that short-term ROCK targeting, or 'priming', improved chemotherapy efficacy, disrupted cancer cell collective movement, and impaired metastasis. This body of work strongly indicates that the use of ROCK inhibitors in pancreatic cancer therapy as 'priming' agents warrants further consideration, and provides insights as to how transient mechanical manipulation, or fine-tuning the ECM, rather than chronic stromal ablation might be beneficial for improving chemotherapeutic efficacy in the treatment of this deadly disease.
Asunto(s)
Terapia Molecular Dirigida/métodos , Neoplasias Pancreáticas/tratamiento farmacológico , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Progresión de la Enfermedad , Humanos , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
The increasingly recognised importance of viscoelastic properties of cells in pathological conditions requires rapid development of advanced cell microrheology technologies. Here, we present a novel Atomic Force Microscopy (AFM)-microrheology (AFM2) method for measuring the viscoelastic properties in living cells, over a wide range of continuous frequencies (0.005 Hz ~ 200 Hz), from a simple stress-relaxation nanoindentation. Experimental data were directly analysed without the need for pre-conceived viscoelastic models. We show the method had an excellent agreement with conventional oscillatory bulk-rheology measurements in gels, opening a new avenue for viscoelastic characterisation of soft matter using minute quantity of materials (or cells). Using this capability, we investigate the viscoelastic responses of cells in association with cancer cell invasive activity modulated by two important molecular regulators (i.e. mutation of the p53 gene and Rho kinase activity). The analysis of elastic (G'(ω)) and viscous (Gâ³(ω)) moduli of living cells has led to the discovery of a characteristic transitions of the loss tangent (Gâ³(ω)/G'(ω)) in the low frequency range (0.005 Hz ~ 0.1 Hz) that is indicative of the capability for cell restructuring of F-actin network. Our method is ready to be implemented in conventional AFMs, providing a simple yet powerful tool for measuring the viscoelastic properties of living cells.
Asunto(s)
Microscopía de Fuerza Atómica/instrumentación , Microscopía de Fuerza Atómica/métodos , Línea Celular Tumoral , Elasticidad , Humanos , ViscosidadRESUMEN
The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries.Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321-36. ©2018 AACR.
Asunto(s)
2-Hidroxifenetilamina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 2-Hidroxifenetilamina/farmacología , 2-Hidroxifenetilamina/uso terapéutico , Amidas/farmacología , Amidas/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral/trasplante , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Invasividad Neoplásica/patología , Invasividad Neoplásica/prevención & control , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/metabolismoRESUMEN
The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKß contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy.Significance: The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. Cancer Res; 78(8); 2096-114. ©2018 AACR.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Descubrimiento de Drogas , Proteína Quinasa de Distrofia Miotónica/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Neoplasias Cutáneas/enzimología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a KrasG12D/p53R172H mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three-dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK-induced genes that facilitate extracellular matrix remodeling, with greatest fold-changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13 MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three-dimensional contexts. Treatment of KrasG12D/p53R172H PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor-associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth.
Asunto(s)
Adenocarcinoma/patología , Carcinoma Ductal Pancreático/patología , Colágeno/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Animales , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Ratones , Análisis de SupervivenciaRESUMEN
The RhoA and RhoC GTPases act via the ROCK1 and ROCK2 kinases to promote actomyosin contraction, resulting in directly induced changes in cytoskeleton structures and altered gene transcription via several possible indirect routes. Elevated activation of the Rho/ROCK pathway has been reported in several diseases and pathological conditions, including disorders of the central nervous system, cardiovascular dysfunctions and cancer. To determine how increased ROCK signalling affected gene expression in pancreatic ductal adenocarcinoma (PDAC) cells, we transduced mouse PDAC cell lines with retroviral constructs encoding fusion proteins that enable conditional activation of ROCK1 or ROCK2, and subsequently performed RNA sequencing (RNA-Seq) using the Illumina NextSeq 500 platform. We describe how gene expression datasets were generated and validated by comparing data obtained by RNA-Seq with RT-qPCR results. Activation of ROCK1 or ROCK2 signalling induced significant changes in gene expression that could be used to determine how actomyosin contractility influences gene transcription in pancreatic cancer.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/genética , Quinasas Asociadas a rho/genética , Adenocarcinoma/metabolismo , Animales , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Neoplasias Pancreáticas/metabolismo , Transducción de Señal , Transfección , Quinasas Asociadas a rho/metabolismoRESUMEN
The serine/threonine kinases ROCK1 and ROCK2 are central mediators of actomyosin contractile force generation that act downstream of the RhoA small GTP-binding protein. As a result, they have key roles in regulating cell morphology and proliferation, and have been implicated in numerous pathological conditions and diseases including hypertension and cancer. Here we describe the generation of a gene-targeted mouse line that enables CRE-inducible expression of a conditionally-active fusion between the ROCK2 kinase domain and the hormone-binding domain of a mutated estrogen receptor (ROCK2:ER). This two-stage system of regulation allows for tissue-selective expression of the ROCK2:ER fusion protein, which then requires administration of estrogen analogues such as tamoxifen or 4-hydroxytamoxifen to elicit kinase activity. This conditional gain-of-function system was validated in multiple tissues by crossing with mice expressing CRE recombinase under the transcriptional control of cytokeratin14 (K14), murine mammary tumor virus (MMTV) or cytochrome P450 Cyp1A1 (Ah) promoters, driving appropriate expression in the epidermis, mammary or intestinal epithelia respectively. Given the interest in ROCK signaling in normal physiology and disease, this mouse line will facilitate research into the consequences of ROCK activation that could be used to complement conditional knockout models. Birth Defects Research (Part A) 106:636-646, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Receptor alfa de Estrógeno/genética , Proteínas Recombinantes de Fusión/genética , Quinasas Asociadas a rho/genética , Animales , Citocromo P-450 CYP1A1/genética , Epidermis/metabolismo , Receptor alfa de Estrógeno/biosíntesis , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Integrasas/genética , Mucosa Intestinal/metabolismo , Glándulas Mamarias Animales/metabolismo , Ratones , Especificidad de Órganos/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Transducción de Señal/efectos de los fármacos , Tamoxifeno/administración & dosificación , Quinasas Asociadas a rho/biosíntesisAsunto(s)
Páncreas , Neoplasias Pancreáticas , Línea Celular Tumoral , Humanos , Células del EstromaRESUMEN
Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt actin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using two independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage-tracing approach we follow the progeny of myoepithelial cells that express α-smooth muscle actin and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.
Asunto(s)
Células Epiteliales/citología , Glándulas Mamarias Animales/citología , Miocitos del Músculo Liso/metabolismo , Células Madre/citología , Actinas/metabolismo , Animales , Benzamidas/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dioxoles/farmacología , Células Epiteliales/metabolismo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Glándulas Mamarias Animales/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Microscopía Fluorescente , Miocitos del Músculo Liso/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismoRESUMEN
The exoRNase Eri1 inhibits RNA interference and trims the 5.8S rRNA 3' end. It also binds to the stem-loop of histone mRNAs, but the functional importance of this interaction remains elusive. Histone mRNAs are normally degraded at the end of S phase or after pharmacological inhibition of replication. Both processes are impaired in Eri1-deficient mouse cells, which instead accumulate oligouridylated histone mRNAs. Eri1 trims the mature histone mRNAs by two unpaired nucleotides at the 3' end but stalls close to the double-stranded stem. Upon oligouridylation of the histone mRNA, the Lsm1-7 heteroheptamer recognizes the oligo(U) tail and interacts with Eri1, whose catalytic activity is then able to degrade the stem-loop in a stepwise manner. These data demonstrate how degradation of histone mRNAs is initiated when 3' oligouridylation creates a cis element that enables Eri1 to process the double-stranded stem-loop structure.
Asunto(s)
Exonucleasas/metabolismo , Histonas/genética , Secuencias Invertidas Repetidas , ARN Mensajero/química , ARN Mensajero/metabolismo , Animales , Biocatálisis , Ciclo Celular , Células Cultivadas , Exonucleasas/genética , Exorribonucleasas , Femenino , Histonas/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Conformación de Ácido Nucleico , Oligorribonucleótidos/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Nucleótidos de Uracilo/metabolismoRESUMEN
The Rho-associated (ROCK) serine/threonine kinases have emerged as central regulators of the actomyosin cytoskeleton, their main purpose being to promote contractile force generation. Aided by the discovery of effective inhibitors such as Y27632, their roles in cancer have been extensively explored with particular attention focused on motility, invasion and metastasis. Recent studies have revealed a surprisingly diverse range of functions of ROCK. These insights could change the way ROCK inhibitors might be used in cancer therapy to include the targeting of stromal rather than tumour cells, the concomitant blocking of ROCK and proteasome activity in K-Ras-driven lung cancers and the combination of ROCK with tyrosine kinase inhibitors for treating haematological malignancies such as chronic myeloid leukaemia. Despite initial optimism for therapeutic efficacy of ROCK inhibition for cancer treatment, no compounds have progressed into standard therapy so far. However, by carefully defining the key cancer types and expanding the appreciation of ROCK's role in cancer beyond being a cell-autonomous promoter of tumour cell invasion and metastasis, the early promise of ROCK inhibitors for cancer therapy might still be realized.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Leucemia Mieloide/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Citoesqueleto/metabolismo , Humanos , Leucemia Mieloide/enzimología , Neoplasias Pulmonares/enzimología , MicroARNs/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/uso terapéutico , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Quinasas Asociadas a rho/química , Quinasas Asociadas a rho/metabolismoRESUMEN
The molecular mechanism by which roquin controls the expression of inducible costimulator (ICOS) to prevent autoimmunity remains unsolved. Here we show that in helper T cells, roquin localized to processing (P) bodies and downregulated ICOS expression. The repression was dependent on the RNA helicase Rck, and roquin interacted with Rck and the enhancer of decapping Edc4, which act together in mRNA decapping. Sequences in roquin that confer P-body localization were essential for roquin-mediated ICOS repression. However, this process did not require microRNAs or the RNA-induced silencing complex (RISC). Instead, roquin bound ICOS mRNA directly, showing an intrinsic preference for a previously unrecognized sequence in the 3' untranslated region (3' UTR). Our results support a model in which roquin controls ICOS expression through binding to the 3' UTR of ICOS mRNA and by interacting with proteins that confer post-transcriptional repression.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , ARN Helicasas DEAD-box/inmunología , MicroARNs/genética , Proteínas Proto-Oncogénicas/inmunología , ARN Mensajero/metabolismo , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Autoinmunidad/genética , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , ARN Helicasas DEAD-box/genética , Regulación de la Expresión Génica , Proteína Coestimuladora de Linfocitos T Inducibles , Ratones , Ratones Mutantes , Ratones Transgénicos , MicroARNs/inmunología , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Mensajero/inmunología , Ubiquitina-Proteína Ligasas/inmunologíaRESUMEN
Eri1 is a 3'-to-5' exoribonuclease conserved from fission yeast to humans. Here we show that Eri1 associates with ribosomes and ribosomal RNA (rRNA). Ribosomes from Eri1-deficient mice contain 5.8S rRNA that is aberrantly extended at its 3' end, and Eri1, but not a catalytically inactive mutant, converts this abnormal 5.8S rRNA to the wild-type form in vitro and in cells. In human and murine cells, Eri1 localizes to the cytoplasm and nucleus, with enrichment in the nucleolus, the site of preribosome biogenesis. RNA binding residues in the Eri1 SAP and linker domains promote stable association with rRNA and thereby facilitate 5.8S rRNA 3' end processing. Taken together, our findings indicate that Eri1 catalyzes the final trimming step in 5.8S rRNA processing, functionally and spatially connecting this regulator of RNAi with the basal translation machinery.