RESUMEN
Protein translation has emerged as a critical bottleneck for overall productivity of biological molecules. An augmentation of protein translation can be achieved by cell line engineering or by sophisticated vector design. However, for industrial process development purposes, identification of media additives that promote translation will be of great value, obviating the generation of new host platforms. Here, we examined the effect of low cadmium chloride concentrations on protein synthesis and cell line productivity. At low micromolar concentrations, cadmium chloride induced the mTOR pathway and promoted total protein synthesis in HEK 293T and CHO-K1 cells with minimal toxicity. In a parallel screening of kinase inhibitors for promoting protein expression, we identified the RSK1 inhibitor, BI-D1870, as having a transcription promoting activity on cytomegalovirus promoter-driven transgenes. Fed-batch analyses of CHO-K1 cells producing the anticoagulant factor tissue plasminogen activator (tPA) demonstrated that inclusion of cadmium chloride alone and particularly in combination with BI-D1870 improved overall yields of tPA by more than two-fold with minimal effect on cell growth. We, therefore, underscore the use of cadmium alone and in combination with BI-D1870 for improving bioproduction yields.
Asunto(s)
Cloruro de Cadmio/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Recombinantes , Animales , Células CHO , Cloruro de Cadmio/toxicidad , Supervivencia Celular/efectos de los fármacos , Cricetulus , Células HEK293 , Humanos , Pteridinas/farmacología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Activador de Tejido Plasminógeno/análisis , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismoRESUMEN
WRN belongs to the RecQ family of DNA helicases and it plays a role in recombination, replication, telomere maintenance and long-patch base excision repair. Here, we demonstrate that WRN efficiently unwinds DNA substrates containing a 1-nucleotide gap in the translocating DNA strand, but when the gap size is increased to 3-nucleotides unwinding activity significantly declines. In contrast, E. coli UvrD (3'-->5' helicase), which recognizes nicks in DNA to initiate unwinding, does not unwind past a 1-nucleotide gap. This unique ability of WRN to bypass gaps supports its involvement in DNA replication and LP-BER where such gaps can be produced by glycosylases and the apurinic/apyrimidinic endonuclease 1 (APE1). Furthermore, we tested telomere repeat binding factor 2 (TRF2), both variants 1 and 2 of protector of telomeres 1 (POT1v1 and POT1v2) and RPA on telomeric DNA substrates containing much bigger gaps than 3-nucleotides in order to determine whether unwinding could be facilitated through WRN-protein interaction. Interestingly, POT1v1 and RPA are capable of stimulating WRN helicase on gapped DNA and 5'-overhang substrates, respectively.
Asunto(s)
Replicación del ADN , Exodesoxirribonucleasas/genética , RecQ Helicasas/genética , Proteína de Replicación A/fisiología , Proteínas de Unión a Telómeros/fisiología , Línea Celular , ADN , Proteínas de Escherichia coli , Humanos , Conformación de Ácido Nucleico , Complejo Shelterina , Helicasa del Síndrome de WernerRESUMEN
Werner syndrome (WS) is a rare autosomal recessive disorder in humans characterized by premature aging and genetic instability. WS is caused by mutations in the WRN gene, which encodes a member of the RecQ family of DNA helicases. Cellular and biochemical studies suggest that WRN plays roles in DNA replication, DNA repair, telomere maintenance, and homologous recombination and that WRN has multiple enzymatic activities including 3' to 5' exonuclease, 3' to 5' helicase, and ssDNA annealing. The goal of this study was to map and further characterize the ssDNA annealing activity of WRN. Enzymatic studies using truncated forms of WRN identified a C-terminal 79 amino acid region between the RQC and the HRDC domains (aa1072-1150) that is required for ssDNA annealing activity. Deletion of the region reduced or eliminated ssDNA annealing activity of the WRN protein. Furthermore, the activity appears to correlate with DNA binding and oligomerization status of the protein.
Asunto(s)
ADN de Cadena Simple/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , RecQ Helicasas/química , RecQ Helicasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , ADN de Cadena Simple/química , Exodesoxirribonucleasas/genética , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , RecQ Helicasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Síndrome de Werner , Helicasa del Síndrome de WernerRESUMEN
BACKGROUND: The Werner protein (WRN), defective in the premature aging disorder Werner syndrome, participates in a number of DNA metabolic processes, and we have been interested in the possible regulation of its function in DNA repair by post-translational modifications. Acetylation mediated by histone acetyltransferases is of key interest because of its potential importance in aging, DNA repair and transcription. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have investigated the p300 acetylation mediated changes on the function of WRN in base excision DNA repair (BER). We show that acetylation of WRN increases in cells treated with methyl methanesulfonate (MMS), suggesting that acetylation of WRN may play a role in response to DNA damage. This hypothesis is consistent with our findings that acetylation of WRN stimulates its catalytic activities in vitro and in vivo, and that acetylated WRN enhances pol beta-mediated strand displacement DNA synthesis more than unacetylated WRN. Furthermore, we show that cellular exposure to the histone deacetylase inhibitor sodium butyrate stimulates long patch BER in wild type cells but not in WRN depleted cells, suggesting that acetylated WRN participates significantly in this process. CONCLUSION/SIGNIFICANCE: Collectively, these results provide the first evidence for a specific role of p300 mediated WRN acetylation in regulating its function during BER.
Asunto(s)
Reparación del ADN , ADN/química , Exodesoxirribonucleasas/química , RecQ Helicasas/química , Envejecimiento , Catálisis , Línea Celular , Daño del ADN , Exodesoxirribonucleasas/metabolismo , Exonucleasas/metabolismo , Células HeLa , Histona Acetiltransferasas/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , RecQ Helicasas/metabolismo , Proteínas Recombinantes/química , Transcripción Genética , Helicasa del Síndrome de WernerRESUMEN
The Cockayne syndrome B (CSB) protein--defective in a majority of patients suffering from the rare autosomal disorder CS--is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP-DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2'-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.
Asunto(s)
ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Línea Celular Transformada , ADN Helicasas/fisiología , Enzimas Reparadoras del ADN/fisiología , Genoma Humano , Humanos , Metilmetanosulfonato/toxicidad , Proteínas de Unión a Poli-ADP-Ribosa , Timidina/análogos & derivados , Timidina/toxicidadRESUMEN
Genome instability is a characteristic of cancer and aging, and is a hallmark of the premature aging disorder Werner syndrome (WS). Evidence suggests that the Werner syndrome protein (WRN) contributes to the maintenance of genome integrity through its involvement in DNA repair. In particular, biochemical evidence indicates a role for WRN in base excision repair (BER). We have previously reported that WRN helicase activity stimulates DNA polymerase beta (pol beta) strand displacement synthesis in vitro. In this report we demonstrate that WRN exonuclease activity can act cooperatively with pol beta, a polymerase lacking 3'-5' proofreading activity. Furthermore, using small interference RNA technology, we demonstrate that WRN knockdown cells are hypersensitive to the alkylating agent methyl methanesulfonate, which creates DNA damage that is primarily repaired by the BER pathway. In addition, repair assays using whole cell extracts from WRN knockdown cells indicate a defect in long patch (LP) BER. These findings demonstrate that WRN plays a direct role in the repair of methylation-induced DNA damage, and suggest a role for both WRN helicase and exonuclease activities together with pol beta during LP BER.
Asunto(s)
ADN Helicasas/fisiología , ADN Polimerasa beta/metabolismo , Reparación del ADN , Exodesoxirribonucleasas/fisiología , Alquilantes/toxicidad , Disparidad de Par Base , Línea Celular , Daño del ADN , ADN Helicasas/antagonistas & inhibidores , Exodesoxirribonucleasas/antagonistas & inhibidores , Humanos , Metilmetanosulfonato/toxicidad , Interferencia de ARN , RecQ Helicasas , Helicasa del Síndrome de WernerRESUMEN
The trk family of receptor tyrosine kinases is crucial for neuronal survival in the vertebrate nervous system, however both C. elegans and Drosophila lack genes encoding trks or their ligands. The only invertebrate representative of this gene family identified to date is Ltrk from the mollusk Lymnaea. Did trophic functions of trk receptors originate early in evolution, or were they an innovation of the vertebrates? Here we show that the Ltrk gene conserves a similar exon/intron order as mammalian trk genes in the region encoding defined extracellular motifs, including one exon encoding a putative variant immunoglobulin-like domain. Chimeric receptors containing the intracellular and transmembrane domains of Ltrk undergo ligand-induced autophosphorylation followed by MAP kinase activation in transfected cells. The chimeras are internalized similarly to TrkA in PC12 cells, and their stimulation leads to differentiation and neurite extension. Knock-down of endogenous Ltrk expression compromises outgrowth and survival of Lymnaea neurons cultured in CNS-conditioned medium. Thus, Ltrk is required for neuronal survival, suggesting that trophic activities of the trk receptor family originated before the divergence of molluscan and vertebrate lineages approximately 600 million years ago.
Asunto(s)
Evolución Biológica , Neuronas/metabolismo , Receptor trkA/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Humanos , Inmunohistoquímica , Hibridación in Situ , Lymnaea , Datos de Secuencia Molecular , Neuronas/citología , Reacción en Cadena de la Polimerasa , Estructura Cuaternaria de Proteína , Receptor trkA/química , Receptor trkA/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de SecuenciaRESUMEN
Trophic survival mechanisms are crucial for the determination of cell numbers in the developing vertebrate nervous system, but important neurotrophic factor families such as the neurotrophins have not yet been found in either Drosophila or C. elegans. Two independent studies on distinct glial populations in Drosophila have now shown that their survival is regulated by EGF family members secreted by adjacent neurons. Fly genetics thus promises new insights on trophic signaling mechanisms and confirms that trophic regulation of cell survival is an evolutionarily ancient mechanism for building the nervous system.