RESUMEN
Structural features and internal dynamics of inosine-containing RNAs are poorly understood. NMR studies of such RNAs require 13 C,15 N-labeling, which cannot be achieved using in vitro transcription as inosine and guanosine are not distinguished by RNA polymerase. Herein, we report the synthesis of an inosine phosphoramidite with selective 13 C8 and 15 N7-isotope incorporation in the base and uniform 13 C-labeling of the ribose. Chemical synthesis of an RNA duplex containing four consecutive IU base pairs with this optimized isotope-labeling scheme greatly simplifies NMR spectra and resolves signal overlap. The absence of detectable NMR signals of imino protons and unusual inter-residue NOE correlations in this RNA indicate deviations from standard A-form geometry, consistent with reduced stability of this duplex seen in UV melting studies compared to its nonedited RNA counterparts. These studies indicate that the introduction of IU base pairs distorts and destabilizes RNA helices significantly compared to the also noncanonical GU base-pairs. Our optimized isotope-labeling scheme enables high-resolution NMR studies of inosine-edited RNAs.
Asunto(s)
Guanosina/química , Inosina/química , Marcaje Isotópico , Compuestos Organofosforados/química , ARN/química , Ribosa/química , Emparejamiento Base , Espectroscopía de Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular , ProtonesRESUMEN
Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA.
Asunto(s)
Antineoplásicos/química , Bisbenzimidazol/química , Bisbenzimidazol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Timidilato Sintasa/genética , Antineoplásicos/farmacología , Disparidad de Par Base , Sitios de Unión , Línea Celular Tumoral , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Modelos Moleculares , ARN Mensajero/química , ARN Mensajero/metabolismo , Timidilato Sintasa/metabolismoRESUMEN
Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene (MECP2), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6. We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.
Asunto(s)
Síndrome de Rett , Humanos , Femenino , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/patología , Neuronas/metabolismo , Histonas/metabolismo , Encéfalo/patología , MutaciónRESUMEN
BACKGROUND: In this study we aimed to describe the morphological and pathogenetic differences between tracheal agenesis and tracheal atresia, which are not clearly distinguished from each other in the literature, and to contribute thereby to the understanding and management of these conditions. Both tracheal agenesis and tracheal atresia represent rare disorders of still unknown aetiology that cannot be detected by prenatal ultrasound. If the affected foetuses survive until birth these conditions result in respiratory failure and in futile attempts to rescue the infant's life. RESULTS: Autopsies and genetic analyses, including singleton or trio exome sequencing, were performed on five neonates/foetuses with tracheal agenesis and three foetuses with tracheal atresia. Tracheal agenesis was characterized by absence of the sublaryngeal trachea and presence of a bronchooesophageal fistula and by pulmonary isomerism and occurred as an isolated malformation complex or as part of a VACTERL association. Special findings were an additional so-called 'pig bronchus' and a first case of tracheal agenesis with sirenomelia. Tracheal atresia presenting with partial obliteration of its lumen and persistence of a fibromuscular streak resulted in CHAOS. This condition was associated with normal lung lobulation and single, non-VACTERL type malformations. Trio ES revealed a novel variant of MAPK11 in one tracheal agenesis case. Its involvement in tracheooesophageal malformation is herein discussed, but remains hypothetical. CONCLUSION: Tracheal agenesis and tracheal atresia represent different disease entities in terms of morphology, pathogenesis and accompanying anomalies due to a primary developmental and secondary disruptive possibly vascular disturbance, respectively.
Asunto(s)
Deformidades Congénitas de las Extremidades , Tráquea/anomalías , Recién Nacido , Embarazo , Femenino , Humanos , Constricción Patológica , Esófago/anomalíasRESUMEN
The transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2) is an intrinsically disordered protein, mutations in which, are implicated in the onset of Rett Syndrome, a severe and debilitating neurodevelopmental disorder. Delivery of this protein fused to the cell-penetrating peptide TAT could allow for the intracellular replenishment of functional MeCP2 and hence potentially serve as a prospective Rett Syndrome therapy. This work outlines the expression, purification and characterization of various TAT-MeCP2 constructs as well as their full-length and shortened eGFP fusion variants. The latter two constructs were used for intracellular uptake studies with subsequent analysis via western blotting and live-cell imaging. All purified MeCP2 samples exhibited high degree of stability and very little aggregation propensity. Full length and minimal TAT-MeCP2-eGFP were found to efficiently transduce into human dermal and murine fibroblasts and localize to cell nuclei. These findings clearly support the utility of MeCP2-based protein replacement therapy as a potential Rett Syndrome treatment option.
Asunto(s)
Proteína 2 de Unión a Metil-CpG , Síndrome de Rett , Animales , Núcleo Celular , Humanos , Proteína 2 de Unión a Metil-CpG/química , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Mutación , Estudios Prospectivos , Síndrome de Rett/genética , Síndrome de Rett/metabolismoRESUMEN
Rett syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants leading to functional impairment of the MeCP2 protein. Here, we used purified recombinant MeCP2e1 and MeCP2e2 protein variants fused to a TAT protein transduction domain (PTD) to evaluate their transduction ability into RTT patient-derived fibroblasts and the ability to carry out their cellular function. We then assessed their transduction ability and therapeutic effects in a RTT mouse model. In vitro, TAT-MeCP2e2-eGFP reversed the pathological hyperacetylation of histones H3K9 and H4K16, a hallmark of abolition of MeCP2 function. In vivo, intraperitoneal administration of TAT-MeCP2e1 and TAT-MeCP2e2 extended the lifespan of Mecp2-/y mice by >50%. This was accompanied by rescue of hippocampal CA2 neuron size in animals treated with TAT-MeCP2e1. Taken together, these findings provide a strong indication that recombinant TAT-MeCP2 can reach mouse brains following peripheral injection and can ameliorate the phenotype of RTT mouse models. Thus, our study serves as a first step in the development of a potentially novel RTT therapy.
Asunto(s)
Síndrome de Rett , Animales , Modelos Animales de Enfermedad , Productos del Gen tat/genética , Productos del Gen tat/uso terapéutico , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Mutación , Fenotipo , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismoRESUMEN
The structure of a cytosine-cytosine (CC) mismatch-containing RNA molecule derived from a hairpin structure in the thymidylate synthase mRNA that binds the aminoglycoside paromomycin with high affinity was determined using nuclear magnetic resonance (NMR) spectroscopy. The cytosines in the mismatch form a noncanonical base pair where both cytosines are uncharged and stack within the stem of the RNA structure. Binding to paromomycin was analyzed using isothermal titration calorimetry (ITC) to demonstrate the necessity of the CC mismatch and to determine the affinity dissociation constant of this RNA to paromomycin to be 0.5 +/- 0.3 microM. The CC mismatch, and the neighboring GC base pairs experienced the highest degree of chemical shift changes in their H6 and H5 resonances indicating that paromomycin binds in the major groove at the CC mismatch site. In comparing the structure of CC mismatch RNA with a fully Watson-Crick GC base paired stem, the CC mismatch is shown to confer a widening of the major groove. This widening, combined with the dynamic nature of the CC mismatch, enables binding of paromomycin to this RNA molecule.
Asunto(s)
Disparidad de Par Base , Conformación de Ácido Nucleico , Paromomicina/metabolismo , ARN Mensajero/metabolismo , Timidilato Sintasa/metabolismo , Sitios de Unión , Calorimetría , Citosina/metabolismo , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.
Asunto(s)
Encéfalo/metabolismo , Mediciones Luminiscentes , Proteína 2 de Unión a Metil-CpG/metabolismo , Animales , Electroquímica , Fibroblastos/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Transporte de ProteínasRESUMEN
Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Proteína 2 de Unión a Metil-CpG/análisis , Proteína 2 de Unión a Metil-CpG/farmacocinética , Animales , Química Encefálica , Células Cultivadas , Técnicas Electroquímicas/métodos , Femenino , Fibroblastos/química , Fibroblastos/metabolismo , Células HEK293 , Humanos , Mediciones Luminiscentes/métodos , Masculino , Proteína 2 de Unión a Metil-CpG/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/farmacocinéticaRESUMEN
Incorporation of the amino acid selenocysteine into a growing protein chain involves the interaction between a hairpin in the mRNA termed the selenocysteine insertion sequence (SECIS) and the special elongation factor SelB. Here we present the structure of the SECIS from the thermophilic organism Moorella thermoacetica (SECIS-MT) determined using nuclear magnetic resonance (NMR) spectroscopy. The SECIS-MT hairpin structure contains a pentaloop with the first and fourth nucleotides of the loop forming a noncanonical GC base pair; the fifth loop nucleotide is bulged out and unstructured. The G and U in positions two and three are on opposite sides of the loop and solvent exposed. The backbone resonances of the SECIS-binding domain from the M. thermoacetica SelB protein were assigned, and the degree of chemical shift perturbations that occur upon SECIS binding were mapped onto the structure of the complex. We demonstrate that a region in the third winged-helix domain of SelB, not previously implicated in binding, is affected by SECIS binding.