RESUMEN
Mutations in p97/VCP cause two motor neuron diseases: inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia and familial amyotrophic lateral sclerosis. How p97 mutations lead to motor neuron degeneration is, however, unknown. Here we used patient-derived induced pluripotent stem cells to generate p97 mutant motor neurons. We reduced the genetic background variation by comparing mutant motor neurons to its isogenic wild type lines. Proteomic analysis reveals that p97R155H/+ motor neurons upregulate several cell cycle proteins at Day 14, but this effect diminishes by Day 20. Molecular changes linked to delayed cell cycle exit are observed in p97 mutant motor neurons. We also find that two p97 inhibitors, CB-5083 and NMS-873, restore some dysregulated protein levels. In addition, two p97 inhibitors and a food and drug administration-approved cyclin-dependent kinase 4/6 inhibitor, Abemaciclib, can rescue motor neuron death. Overall, we successfully used iPSC-derived motor neurons, identified dysregulated proteome and transcriptome and showed that p97 inhibitors rescue phenotypes in this disease model.
RESUMEN
In many organisms, the circadian clock is composed of functionally coupled morning and evening oscillators. In Arabidopsis, oscillator coupling relies on a core loop in which the evening oscillator component TIMING OF CAB EXPRESSION 1 (TOC1) was proposed to activate a subset of morning-expressed oscillator genes. Here, we show that TOC1 does not function as an activator but rather as a general repressor of oscillator gene expression. Repression occurs through TOC1 rhythmic association to the promoters of the oscillator genes. Hormone-dependent induction of TOC1 and analysis of RNA interference plants show that TOC1 prevents the activation of morning-expressed genes at night. Our study overturns the prevailing model of the Arabidopsis circadian clock, showing that the morning and evening oscillator loops are connected through the repressing activity of TOC1.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Relojes Circadianos , Regulación de la Expresión Génica de las Plantas , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Inmunoprecipitación de Cromatina , Relojes Circadianos/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Oscuridad , Dexametasona/farmacología , Retroalimentación Fisiológica , Genes de Plantas , Luz , Fotoperiodo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Interferencia de ARN , Proteínas Represoras/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The mitochondrial HMG-box transcription factor xl-mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl-mtTFA binding to this site. First, co-immunoprecipitation of differentially tagged recombinant mtTFA derivatives established that the protein exists as a multimer. Second, in vitro chemical cross-linking experiments provided evidence of cross-linked dimers, trimers and tetramers of xl-mtTFA. Finally, high resolution scanning transmission electron microscopy (STEM) established that xl-mtTFA binds to the specific promoter-proximal site predominantly as a tetramer. Computer analysis of several previously characterized binding sites for xl-mtTFA revealed a fine structure consisting of two half-sites in a symmetrical orientation. The predominant sequence of this dyad symmetry motif shows homology to binding sites of sequence-specific HMG-box-containing proteins such as Sry and Lef-1. We suggest that bidirectional activation of transcription results from the fact that binding of a tetramer of xl-mtTFA permits symmetrical interactions with other components of the transcription machinery at the adjacent core promoters.
Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Mitocondrias/genética , Transactivadores/metabolismo , Activación Transcripcional , Proteínas de Xenopus , Animales , Sitios de Unión , Secuencia de Consenso , Reactivos de Enlaces Cruzados , ADN Mitocondrial/efectos de los fármacos , ADN Mitocondrial/ultraestructura , Glutaral/farmacología , Proteínas del Grupo de Alta Movilidad/efectos de los fármacos , Proteínas del Grupo de Alta Movilidad/ultraestructura , Microscopía Electrónica de Rastreo , Nucleoproteínas/ultraestructura , Pruebas de Precipitina , Unión Proteica , Conformación Proteica , Transactivadores/efectos de los fármacos , Transactivadores/ultraestructura , Xenopus laevisRESUMEN
Transcription of Xenopus laevis mitochondrial DNA (xl-mtDNA) by the mitochondrial RNA polymerase requires a dissociable factor. This factor was purified to near homogeneity and identified as a 40-kDa protein. A second protein implicated in the transcription of mtDNA, the Xenopus homolog of the HMG box protein mtTFA, was also purified to homogeneity and partially sequenced. The sequence of a cDNA clone encoding xl-mtTFA revealed a high degree of sequence similarity to human and Saccharomyces cerevisiae mtTFA. xl-mtTFA was not required for basal transcription from a minimal mtDNA promoter, and this HMG box factor could not substitute for the basal factor, which is therefore designated xl-mtTFB. An antibody directed against the N terminus of xl-mtTFA did not cross-react with xl-mtTFB. xl-mtTFA is an abundant protein that appears to have at least two functions in mitochondria. First, it plays a major role in packaging mtDNA within the organelle. Second, DNase I footprinting experiments identified preferred binding sites for xl-mtTFA within the control region of mtDNA next to major mitochondrial promoters. We show that binding of xl-mtTFA to a site separating the two clusters of bidirectional promoters selectively stimulates specific transcription in vitro by the basal transcription machinery, comprising mitochondrial RNA polymerase and xl-mtTFB.
Asunto(s)
ADN Mitocondrial/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Mitocondrias/enzimología , Transactivadores , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Femenino , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Moldes Genéticos , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Xenopus laevisRESUMEN
The 8-oxoguanine-DNA glycosylase of Escherichia coli, also known as formamidopyrimidine-DNA glycosylase (Fpg protein), has N-glycosylase and AP-lyase activities. This enzyme repairs oxidative DNA damage by efficiently removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA. Defined oligodeoxynucleotides containing various 8-oxopurines were used to examine the substrate specificity of Fpg protein and to establish the role of functional groups in DNA on damage recognition and catalysis. Binding affinities of Fpg protein were established for duplex oligodeoxynucleotides containing 8-oxo-2'-deoxyguanine, 8-oxo-2'-deoxyadenine, 8-oxo-2'-deoxynebularine, 8-oxo-2'-deoxyinosine, abasic sites, and a ring-open adduct of C8-aminofluorene guanine. The C8 keto group of 8-oxodG:dC presents in the major groove and is correlated with tight binding (Kd = 8.9 nM). Binding is much weaker when the C8 keto functional group is in the minor groove, as in 8-oxodG:dA (Kd = 340 nM). Km and Vmax were determined for the cleavage reaction. Specificity constants (Kcat/Km) are consistently higher for oligodeoxynucleotide duplexes containing 8-oxopurines with C6 and C8 keto groups, as in 8-oxodG:dC and 8-oxodI:dC, where Kcat/Km are 9.3 and 18 min-1 nM x 10(-3), respectively. 8-oxodN:dC lacks the C6 keto group; the specificity constant is 0.024 min-1 nM x 10(-3). Taken together, our data suggest that the C8 keto group of 8-oxodeoxyguanine and the carbonyl moiety of formamidopyrimidine enable Fpg protein to recognize and bind duplex DNA containing these modified bases. An enzyme-catalyzed reaction involving the C6 keto group of the substrate leads to removal of these lesions. A mechanism involving protonation at O-6 of 8-oxoguanine is proposed to account for the N-glycosylase activity of this enzyme.