RESUMEN
Protein arginine methyltransferase 5 (PRMT5) complexed with MEP50/WDR77 catalyzes arginine methylation on histones and other proteins. PRMT5-MEP50 activity is elevated in cancer cells and its expression is highly correlated with poor prognosis in many human tumors. We demonstrate that PRMT5-MEP50 is essential for transcriptional regulation promoting cancer cell invasive phenotypes in lung adenocarcinoma, lung squamous cell carcinoma and breast carcinoma cancer cells. RNA-Seq transcriptome analysis demonstrated that PRMT5 and MEP50 are required to maintain expression of metastasis and Epithelial-to-mesenchymal transition (EMT) markers and to potentiate an epigenetic mechanism of the TGFß response. We show that PRMT5-MEP50 activity both positively and negatively regulates expression of a wide range of genes. Exogenous TGFß promotes EMT in a unique pathway of PRMT5-MEP50 catalyzed histone mono- and dimethylation of chromatin at key metastasis suppressor and EMT genes, defining a new mechanism regulating cancer invasivity. PRMT5 methylation of histone H3R2me1 induced transcriptional activation by recruitment of WDR5 and concomitant H3K4 methylation at targeted genes. In parallel, PRMT5 methylation of histone H4R3me2s suppressed transcription at distinct genomic loci. Our decoding of histone methylarginine at key genes supports a critical role for complementary PRMT5-MEP50 transcriptional activation and repression in cancer invasion pathways and in response to TGFß stimulation and therefore orients future chemotherapeutic opportunities.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Perfilación de la Expresión Génica/métodos , Histonas/metabolismo , Neoplasias/patología , Proteína-Arginina N-Metiltransferasas/genética , Análisis de Secuencia de ARN/métodos , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Arginina/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Epigénesis Genética , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Células MCF-7 , Metilación , Invasividad Neoplásica , Neoplasias/genética , Pronóstico , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Minichromosome maintenance proteins (MCMs) form a family of conserved molecules that are essential for initiation of DNA replication. All eukaryotes contain six orthologous MCM proteins that function as heteromultimeric complexes. The sequencing of the complete genomes of several archaebacteria has shown that MCM proteins are also present in archaea. The archaea Methanobacterium thermoautotrophicum contains a single MCM-related sequence. Here we report on the expression and purification of the recombinant M. thermoautotrophicum MCM protein (MtMCM) in both Escherichia coli and baculovirus-infected cells. We show that purified MtMCM protein assembles in large macromolecular complexes consistent in size with being double hexamers. We demonstrate that MtMCM contains helicase activity that preferentially uses dATP and DNA-dependent dATPase and ATPase activities. The intrinsic helicase activity of MtMCM is abolished when a conserved lysine in the helicase domain I/nucleotide binding site is mutated. MtMCM helicase unwinds DNA duplexes in a 3' --> 5' direction and can unwind up to 500 base pairs in vitro. The kinetics, processivity, and directionality of MtMCM support its role as a replicative helicase in M. thermoautotrophicum. This strongly suggests that this function is conserved for MCM proteins in eukaryotes where a replicative helicase has yet to be identified.
Asunto(s)
Proteínas Arqueales/metabolismo , ADN Helicasas/metabolismo , Methanobacterium/genética , Methanobacterium/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Clonación Molecular , Secuencia Conservada , Replicación del ADN , Escherichia coli , Cinética , Lisina , Sustancias Macromoleculares , Methanobacterium/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Spodoptera , Especificidad por Sustrato , TransfecciónRESUMEN
BACKGROUND: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ("sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the beta subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. RESULTS: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd approximately 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 approximately 24 min). beta is more stable in solution (Kd < 60 PM) and on DNA (t1/2 approximately 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd approximately 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and beta clamps on DNA necessitates an active process for their removal. The clamp loaders (RFC and gamma complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. CONCLUSIONS: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and beta clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and beta double as clamp unloaders presumably for the purpose of clamp recycling.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Bacteriófago T4/metabolismo , ADN/metabolismo , Replicación del ADN , Estabilidad de Enzimas , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Conformación Proteica , SolucionesAsunto(s)
Hallux Valgus/diagnóstico por imagen , Adolescente , Adulto , Anciano , Niño , Femenino , Hallux Valgus/cirugía , Humanos , Masculino , Persona de Mediana Edad , Osteotomía , RadiografíaRESUMEN
The presence of a postoperative wound infection after clean podiatric surgery may predispose the patient to a prolonged, often nonambulatory, convalescent period. Although the incidence of postoperative infection after elective podiatric surgery is small, a protocol to govern the use of prophylactic antibiotics has been developed in order to achieve their maximum benefits with a minimum of risk. Indications for chemoprophylaxis have been divided into two groups: specific operative procedures that may warrant antibiotic intervention and patients who present with specific risk factors. Additionally, it has been suggested that single dose prophylaxis is preferable to multiple dose administration.