RESUMEN
Acute myeloid leukemia (AML) cells have high oxidative phosphorylation and mitochondrial mass and low respiratory chain spare reserve capacity. We reasoned that targeting the mitochondrial RNA polymerase (POLRMT), which indirectly controls oxidative phosphorylation, represents a therapeutic strategy for AML. POLRMT-knockdown OCI-AML2 cells exhibited decreased mitochondrial gene expression, decreased levels of assembled complex I, decreased levels of mitochondrially-encoded Cox-II and decreased oxidative phosphorylation. POLRMT-knockdown cells exhibited an increase in complex II of the electron transport chain, a complex comprised entirely of subunits encoded by nuclear genes, and POLRMT-knockdown cells were resistant to a complex II inhibitor theonyltrifluoroacetone. POLRMT-knockdown cells showed a prominent increase in cell death. Treatment of OCI-AML2 cells with 10-50 µM 2-C-methyladenosine (2-CM), a chain terminator of mitochondrial transcription, reduced mitochondrial gene expression and oxidative phosphorylation, and increased cell death in a concentration-dependent manner. Treatment of normal human hematopoietic cells with 2-CM at concentrations of up to 100 µMdid not alter clonogenic growth, suggesting a therapeutic window. In an OCI-AML2 xenograft model, treatment with 2-CM (70 mg/kg, i.p., daily) decreased the volume and mass of tumours to half that of vehicle controls. 2-CM did not cause toxicity to major organs. Overall, our results in a preclinical model contribute to the functional validation of the utility of targeting the mitochondrial RNA polymerase as a therapeutic strategy for AML.
Asunto(s)
Adenosina/análogos & derivados , Antineoplásicos/farmacología , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Adenosina/farmacología , Animales , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Relación Dosis-Respuesta a Droga , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Ratones SCID , Mitocondrias/enzimología , Mitocondrias/patología , Terapia Molecular Dirigida , Fosforilación Oxidativa , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacos , Células U937 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiPARP/ARTD14) is a member of the PARP family and is regulated by the aryl hydrocarbon receptor (AHR); however, little is known about TiPARP function. In this study, we examined the catalytic function of TiPARP and determined its role in AHR transactivation. We observed that TiPARP exhibited auto-mono-ADP-ribosyltransferase activity and ribosylated core histones. RNAi-mediated knockdown of TiPARP in T-47D breast cancer and HuH-7 hepatoma cells increased TCDD-dependent cytochrome P450 1A1 (CYP1A1) and CYP1B1 messenger RNA (mRNA) expression levels and recruitment of AHR to both genes. Overexpression of TiPARP reduced AHR-dependent increases in CYP1A1-reporter gene activity, which was restored by overexpression of AHR, but not aryl hydrocarbon receptor nuclear translocator. Deletion and mutagenesis studies showed that TiPARP-mediated inhibition of AHR required the zinc-finger and catalytic domains. TiPARP and AHR co-localized in the nucleus, directly interacted and both were recruited to CYP1A1 in response to TCDD. Overexpression of Tiparp enhanced, whereas RNAi-mediated knockdown of TiPARP reduced TCDD-dependent AHR proteolytic degradation. TCDD-dependent induction of AHR target genes was enhanced in Tiparp(-/-) mouse embryonic fibroblasts compared with wildtype controls. Our findings show that TiPARP is a mono-ADP-ribosyltransferase and a transcriptional repressor of AHR, revealing a novel negative feedback loop in AHR signalling.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Proteínas Represoras/metabolismo , Activación Transcripcional , ADP Ribosa Transferasas/antagonistas & inhibidores , ADP Ribosa Transferasas/química , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Dominio Catalítico , Línea Celular Tumoral , Núcleo Celular/química , Núcleo Celular/metabolismo , Humanos , Ratones , Ratones Noqueados , Proteínas de Transporte de Nucleósidos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/química , Dibenzodioxinas Policloradas/farmacología , Receptores de Hidrocarburo de Aril/análisis , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Transducción de Señal , Dedos de ZincRESUMEN
Neurodevelopmental defects are observed in the hereditary disorder Cockayne syndrome (CS). The gene most frequently mutated in CS, Cockayne Syndrome B (CSB), is required for the repair of bulky DNA adducts in transcribed genes during transcription-coupled nucleotide excision repair. CSB also plays a role in chromatin remodeling and mitochondrial function. The role of CSB in neural development is poorly understood. Here we report that the abundance of neural progenitors is normal in Csb(-/-) mice and the frequency of apoptotic cells in the neurogenic niche of the adult subependymal zone is similar in Csb(-/-) and wild type mice. Both embryonic and adult Csb(-/-) neural precursors exhibited defective self-renewal in the neurosphere assay. In Csb(-/-) neural precursors, self-renewal progressively decreased in serially passaged neurospheres. The data also indicate that Csb and the nucleotide excision repair protein Xpa preserve embryonic neural stem cell self-renewal after UV DNA damage. Although Csb(-/-) neural precursors do not exhibit altered neuronal lineage commitment after low-dose UV (1J/m(2)) in vitro, neurons differentiated in vitro from Csb(-/-) neural precursors that had been irradiated with 1J/m(2) UV exhibited defective neurite outgrowth. These findings identify a function for Csb in neural precursors.
Asunto(s)
Enzimas Reparadoras del ADN/genética , Células-Madre Neurales/citología , Animales , Apoptosis/genética , Apoptosis/efectos de la radiación , Proliferación Celular , Daño del ADN , Epéndimo/citología , Ratones , Ratones Noqueados , Células-Madre Neurales/efectos de la radiación , Neurogénesis/genética , Neurogénesis/efectos de la radiación , Proteínas de Unión a Poli-ADP-Ribosa , Rayos Ultravioleta , Proteína de la Xerodermia Pigmentosa del Grupo A/genéticaRESUMEN
We present an integrated experimental and computational study of the molecular mechanisms by which myristoylation affects protein folding and function, which has been little characterized to date. Myristoylation, the covalent linkage of a hydrophobic C14 fatty acyl chain to the N-terminal glycine in a protein, is a common modification that plays a critical role in vital regulated cellular processes by undergoing reversible energetic and conformational switching. Coarse-grained folding simulations for the model pH-dependent actin- and membrane-binding protein hisactophilin reveal that nonnative hydrophobic interactions of the myristoyl with the protein as well as nonnative electrostatic interactions have a pronounced effect on folding rates and thermodynamic stability. Folding measurements for hydrophobic residue mutations of hisactophilin and atomistic simulations indicate that the nonnative interactions of the myristoyl group in the folding transition state are nonspecific and robust, and so smooth the energy landscape for folding. In contrast, myristoyl interactions in the native state are highly specific and tuned for sensitive control of switching functionality. Simulations and amide hydrogen exchange measurements provide evidence for increases as well as decreases in stability localized on one side of the myristoyl binding pocket in the protein, implicating strain and altered dynamics in switching. The effects of folding and function arising from myristoylation are profoundly different from the effects of other post-translational modifications.