RESUMEN
In RNA silencing, small RNAs produced by the RNase-III Dicer guide Argonaute-like proteins as part of RNA-induced silencing complexes (RISC) to regulate gene expression transcriptionally or post-transcriptionally. Here, we have characterized the RNA silencing machinery and exhaustive small RNAome of Toxoplasma gondii, member of the Apicomplexa, a phylum of animal- and human-infecting parasites that cause extensive health and economic damages to human populations worldwide. Remarkably, the small RNA-generating machinery of Toxoplasma is phylogenetically and functionally related to that of plants and fungi, and accounts for an exceptionally diverse array of small RNAs. This array includes conspicuous populations of repeat-associated small interfering RNA (siRNA), which, as in plants, likely generate and maintain heterochromatin at DNA repeats and satellites. Toxoplasma small RNAs also include many microRNAs with clear metazoan-like features whose accumulation is sometimes extremely high and dynamic, an unexpected finding given that Toxoplasma is a unicellular protist. Both plant-like heterochromatic small RNAs and metazoan-like microRNAs bind to a single Argonaute protein, Tg-AGO. Toxoplasma miRNAs co-sediment with polyribosomes, and thus, are likely to act as translational regulators, consistent with the lack of catalytic residues in Tg-AGO. Mass spectrometric analyses of the Tg-AGO protein complex revealed a common set of virtually all known RISC components so far characterized in human and Drosophila, as well as novel proteins involved in RNA metabolism. In agreement with its loading with heterochromatic small RNAs, Tg-AGO also associates substoichiometrically with components of known chromatin-repressing complexes. Thus, a puzzling patchwork of silencing processor and effector proteins from plant, fungal and metazoan origin accounts for the production and action of an unsuspected variety of small RNAs in the single-cell parasite Toxoplasma and possibly in other apicomplexans. This study establishes Toxoplasma as a unique model system for studying the evolution and molecular mechanisms of RNA silencing among eukaryotes.
Asunto(s)
Evolución Molecular , Interferencia de ARN/fisiología , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Toxoplasma/genética , Toxoplasmosis/parasitología , Células Cultivadas , Fibroblastos/citología , Fibroblastos/parasitología , Regulación de la Expresión Génica , Genoma de Protozoos , Humanos , MicroARNs/genética , Filogenia , Proteómica , Toxoplasma/crecimiento & desarrolloRESUMEN
Protein kinase CK2 is an unfavorable pronostic marker in several cancers and has consequently emerged as a relevant therapeutic target. Several classes of ATP-competitive inhibitors have been identified, showing variable effectiveness. The molecular architecture of this multisubunit enzyme could offer alternative strategies to develop small molecule inhibitors targeting different surfaces of the kinase. Polyoxometalates were identified as original CK2 inhibitors targeting key structural elements located outside the active site. In addition, the CK2 subunit interface represents an exosite distinct from the catalytic cavity that can be targeted by peptides or small molecules to achieve functional effects.
Asunto(s)
Biomarcadores de Tumor/antagonistas & inhibidores , Quinasa de la Caseína II/antagonistas & inhibidores , Neoplasias/enzimología , Inhibidores de la Síntesis de la Proteína/química , Compuestos de Tungsteno/química , Animales , Biomarcadores de Tumor/química , Biomarcadores de Tumor/metabolismo , Quinasa de la Caseína II/química , Quinasa de la Caseína II/metabolismo , Dominio Catalítico , Humanos , Neoplasias/tratamiento farmacológico , Inhibidores de la Síntesis de la Proteína/uso terapéutico , Compuestos de Tungsteno/uso terapéuticoRESUMEN
Protein kinase casein kinase 2 (CK2) is a serine/threonine kinase with evidence of implication in growth dysregulation and apoptosis resistance, making it a relevant target for cancer therapy. Several CK2 inhibitors have been developed showing variable efficiency, emphasizing the need to expand the chemical diversity of those inhibitors. We report the identification and characterization of 2,8-difurandicarboxylic acid derivatives as a new class of nanomolar ATP-competitive inhibitors. Selectivity profiling pointed out proviral insertion Moloney virus kinases (Pim kinases) as the only other kinases that are significantly inhibited. By combining structure-activity relationship analysis with structural determination, we were able to determine the binding mode of these inhibitors for both kinases and to explain their strong inhibitory potency. Essential chemical features necessary for activity on both kinases were then identified. The described compounds are not cell permeable: however, they could provide a lead for developing novel inhibitors usable also in vivo. Given the similar but not redundant pathophysiological functions of CK2 and Pim family members, such inhibitors would provide new attractive leads for targeted cancer therapy. This work highlights that 2 functionally related kinases from different kinome branches display exquisite sensitivity to a common inhibitor.
Asunto(s)
Quinasa de la Caseína II/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Animales , Sitios de Unión , Quinasa de la Caseína II/química , Línea Celular Tumoral , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Estabilidad de Enzimas , Humanos , Proteínas Proto-Oncogénicas c-pim-1/químicaRESUMEN
The ability of living cells to alter their gene expression patterns in response to environmental changes is essential for viability. Oxidative stress represents a common threat for all aerobic life. In normally growing cells, in which hydrogen peroxide generation is transient or pulsed, the antioxidant systems efficiently control its concentration. Intracellular parasites must also protect themselves against the oxidative burst imposed by the host. In this work, we have investigated the role of KMTox, a new histone lysine methyltransferase, in the obligate intracellular parasite Toxoplasma gondii. KMTox is a nuclear protein that holds a High Mobility Group domain, which is thought to recognize bent DNA. The enzyme methylates both histones H4 and H2A in vitro with a great preference for the substrate in reduced conditions. Importantly, KMTox interacts specifically with the typical 2-cys peroxiredoxin-1 and the binding is to some extent enhanced upon oxidation. It appears that the cellular functions that are primarily regulated by the KMTox are antioxidant defences and maintenance of cellular homeostasis. KMTox may regulate gene expression in T. gondii by providing the rapid re-arrangement of chromatin domains and by interacting with the redox-sensor TgPrx1 contribute to establish the antioxidant 'firewall' in T. gondii.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Estrés Oxidativo , Peroxirredoxinas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Animales , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Peróxido de Hidrógeno/farmacología , Proteínas Protozoarias/genética , ARN Protozoario/genética , Toxoplasma/efectos de los fármacos , Toxoplasma/genéticaRESUMEN
Posttranslational histone modifications modulate chromatin-templated processes in various biological systems. H4K20 methylation is considered to have an evolutionarily ancient role in DNA repair and genome integrity, while its function in heterochromatin function and gene expression is thought to have arisen later during evolution. Here, we identify and characterize H4K20 methylases of the Set8 family in Plasmodium and Toxoplasma, two medically important members of the protozoan phylum Apicomplexa. Remarkably, parasite Set8-related proteins display H4K20 mono-, di-, and trimethylase activities, in striking contrast to the monomethylase-restricted human Set8. Structurally, few residues forming the substrate-specific channel dictate enzyme methylation multiplicity. These enzymes are cell cycle regulated and focally enriched at pericentric and telomeric heterochromatin in both parasites. Collectively, our findings provide new insights into the evolution of Set8-mediated biochemical pathways, suggesting that the heterochromatic function of the marker is not restricted to metazoans. Thus, these lower eukaryotes have developed a diverse panel of biological stages through their high capacity to differentiate, and epigenetics only begins to emerge as a strong determinant of their biology.
Asunto(s)
Silenciador del Gen , Genoma de Protozoos/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/genética , Secuencia de Aminoácidos , Aminoácidos/genética , Animales , Catálisis , Dominio Catalítico , Ciclo Celular , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Parásitos/citología , Parásitos/enzimología , Parásitos/genética , Filogenia , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Toxoplasma/citología , Toxoplasma/enzimologíaRESUMEN
CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2ß subunits is essential for substrate selectivity. The CK2α/CK2ß interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2ß interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.
Asunto(s)
Quinasa de la Caseína II/antagonistas & inhibidores , Holoenzimas/metabolismo , Inhibidores de Proteínas Quinasas/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Quinasa de la Caseína II/metabolismo , Dominio Catalítico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Holoenzimas/química , Humanos , Cinética , Simulación del Acoplamiento Molecular , Péptidos/química , Péptidos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
INTRODUCTION: Due to its physiological role into promoting cell survival and its dysregulation in most cancer cells, protein kinase CK2 is a relevant physiopathological target for development of chemical inhibitors. We report the discovery of azonaphthalene derivatives, as a new family of highly specific CK2 inhibitors. First, we demonstrated that CK2 inhibition (IC50= 0.4 µM) was highly specific, reversible and non ATP-competitive. Small Angle X-ray Scattering experiments showed that this inhibition was due to large conformational change of CK2α upon binding of these inhibitors. We showed that several compounds of the family were cell-potent CK2 inhibitors promoting cell cycle arrest of human glioblastoma U373 cells. Finally, in vitro and in vivo assays showed that these compounds could decrease U373 cell tumor mass by 83 % emphasizing their efficacy against these apoptosis-resistant tumors. In contrast, Azonaphthalene derivatives inactive on CK2 activity showed no effect in colony formation and tumor regression assays. These findings illustrate the emergence of nonclassical CK2 inhibitors and provide exciting opportunities for the development of novel allosteric CK2 inhibitors. BACKGROUND: CK2 is an emerging therapeutic target and ATP-competitive inhibitors have been identiï¬ed. CK2 is endowed with specific structural features providing alternative strategies for inhibition. RESULTS: Azonaphthalene compounds are allosteric CK2 inhibitors showing antitumor activity. CONCLUSION: CK2 may be targeted allosterically. SIGNIFICANCE: These inhibitors provide a foundation for a new paradigm for specific CK2 inhibition.
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos Azo/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Puntos de Control del Ciclo Celular/efectos de los fármacos , Naftalenos/farmacología , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular , Femenino , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Desnudos , Neoplasias/patología , Relación Estructura-ActividadRESUMEN
Protein kinase CK2 (formerly casein kinase 2) is recognized as a central component in the control of the cellular homeostasis; however, much remains unknown regarding its regulation and its implication in cellular transformation and carcinogenesis. Moreover, study of CK2 function and regulation in a cellular context is complicated by the dynamic multisubunit architecture of this protein kinase. Although a number of robust techniques are available to assay CK2 activity in vitro, there is a demand for sensitive and specific assays to evaluate its activity in living cells. We hereby provide a detailed description of several assays for monitoring the CK2 activity and its subunit interaction in living cells. The guidelines presented herein should enable researchers in the field to establish strategies for cellular screenings of CK2 inhibitors.
Asunto(s)
Quinasa de la Caseína II/análisis , Quinasa de la Caseína II/metabolismo , Animales , Quinasa de la Caseína II/genética , Línea Celular , Humanos , Microscopía Fluorescente/métodos , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia/métodos , Transfección/métodosRESUMEN
The alkyloid compound ellipticine derived from the berrywood tree is a topoisomerase II poison that is used in ovarian and breast cancer treatment. In this study, we report the identification of ellipticine derivatives and their tetracyclic angular benzopyridoindole analogues as novel ATP-competitive inhibitors of the protein kinase CK2. In vitro and in vivo assays showed that these compounds have a good pharmacologic profile, causing a marked inhibition of CK2 activity associated with cell cycle arrest and apoptosis in human cancer cells. Further, in vivo assays demonstrate antitumor activity in a mouse xenograft model of human glioblastoma. Finally, crystal structures of CK2-inhibitor complex provide structural insights on the molecular basis of CK2 inhibition. Our work lays the foundation for development of clinically useful CK2 inhibitors derived from a well-studied scaffold with suitable pharmacokinetics parameters.