RESUMEN
Cutaneous melanoma is the most dangerous and deadly form of human skin malignancy. Despite its rarity, it accounts for a staggering 80% of deaths attributed to cutaneous cancers overall. Moreover, its final stages often exhibit resistance to drug treatments, resulting in unfavorable outcomes. Hence, ensuring access to novel and improved chemotherapeutic agents is imperative for patients grappling with this severe ailment. Pyrazole and its fused systems derived thereof are heteroaromatic moieties widely employed in medicinal chemistry to develop effective drugs for various therapeutic areas, including inflammation, pain, oxidation, pathogens, depression, and fever. In a previous study, we described the biochemical properties of a newly synthesized group of imidazo-pyrazole compounds. In this paper, to improve our knowledge of the pharmacological properties of these molecules, we conduct a differential proteomic analysis on a human melanoma cell line treated with one of these imidazo-pyrazole derivatives. Our results detail the changes to the SKMEL-28 cell line proteome induced by 24, 48, and 72 h of 3e imidazo-pyrazole treatment. Notably, we highlight the down-regulation of the Ras-responsive element binding protein 1 (RREB1), a member of the zinc finger transcription factors family involved in the tumorigenesis of melanoma. RREB1 is a downstream element of the MAPK pathway, and its activation is mediated by ERK1/2 through phosphorylation.
Asunto(s)
Melanoma , Proteómica , Pirazoles , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Pirazoles/farmacología , Pirazoles/química , Proteómica/métodos , Línea Celular Tumoral , Factores de Transcripción/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Proteínas de Unión al ADN/metabolismo , Imidazoles/farmacología , Imidazoles/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteoma/metabolismoRESUMEN
Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines and can bind two different receptors, Leukemia inhibitory factor receptor (LIFR) and Oncostatin M receptor (OSMR), through a complex containing the common glycoprotein 130 (gp130) subunit [...].
Asunto(s)
Citocinas , Interleucina-6 , Receptor gp130 de Citocinas/metabolismo , Interleucina-6/metabolismo , Oncostatina M/metabolismo , Receptores de Citocinas/metabolismo , Receptores OSM-LIF , Receptores de Oncostatina M/metabolismoRESUMEN
Glial cells play important roles in the development and homeostasis of metazoan nervous systems. However, while their involvement in the development and function in the central nervous system (CNS) of vertebrates is increasingly well understood, much less is known about invertebrate glia and the evolutionary history of glial cells more generally. An investigation into amphioxus glia is therefore timely, as this organism is the best living proxy for the last common ancestor of all chordates, and hence provides a window into the role of glial cell development and function at the transition of invertebrates and vertebrates. We report here our findings on amphioxus glia as characterized by molecular probes correlated with anatomical data at the transmission electron microscopy (TEM) level. The results show that amphioxus glial lineages express genes typical of vertebrate astroglia and radial glia, and that they segregate early in development, forming what appears to be a spatially separate cell proliferation zone positioned laterally, between the dorsal and ventral zones of neural cell proliferation. Our study provides strong evidence for the presence of vertebrate-type glial cells in amphioxus, while highlighting the role played by segregated progenitor cell pools in CNS development. There are implications also for our understanding of glial cells in a broader evolutionary context, and insights into patterns of precursor cell deployment in the chordate nerve cord.
Asunto(s)
Anfioxos , Animales , Evolución Biológica , Anfioxos/genética , Neurogénesis/fisiología , Neuroglía , VertebradosRESUMEN
PURPOSE: CCHS is an extremely rare congenital disorder requiring artificial ventilation as life support. Typically caused by heterozygous polyalanine repeat expansion mutations (PARMs) in the PHOX2B gene, identification of a relationship between PARM length and phenotype severity has enabled anticipatory management. However, for patients with non-PARMs in PHOX2B (NPARMs, ~10% of CCHS patients), a genotype-phenotype correlation has not been established. This comprehensive report of PHOX2B NPARMs and associated phenotypes, aims at elucidating potential genotype-phenotype correlations that will guide anticipatory management. METHODS: An international collaboration (clinical, commercial, and research laboratories) was established to collect/share information on novel and previously published PHOX2B NPARM cases. Variants were categorized by type and gene location. Categorical data were analyzed with chi-square and Fisher's exact test; further pairwise comparisons were made on significant results. RESULTS: Three hundred two individuals with PHOX2B NPARMs were identified, including 139 previously unreported cases. Findings demonstrate significant associations between key phenotypic manifestations of CCHS and variant type, location, and predicted effect on protein function. CONCLUSION: This study presents the largest cohort of PHOX2B NPARMs and associated phenotype data to date, enabling genotype-phenotype studies that will advance personalized, anticipatory management and help elucidate pathological mechanisms. Further characterization of PHOX2B NPARMs demands longitudinal clinical follow-up through international registries.
Asunto(s)
Genes Homeobox , Proteínas de Homeodominio , Estudios de Asociación Genética , Proteínas de Homeodominio/genética , Humanos , Hipoventilación/congénito , Mutación , Apnea Central del SueñoRESUMEN
The SARS-CoV-2 infection determines the COVID-19 syndrome characterized, in the worst cases, by severe respiratory distress, pulmonary and cardiac fibrosis, inflammatory cytokine release, and immunosuppression. This condition has led to the death of about 2.15% of the total infected world population so far. Among survivors, the presence of the so-called persistent post-COVID-19 syndrome (PPCS) is a common finding. In COVID-19 survivors, PPCS presents one or more symptoms: fatigue, dyspnea, memory loss, sleep disorders, and difficulty concentrating. In this study, a cohort of 117 COVID-19 survivors (post-COVID-19) and 144 non-infected volunteers (COVID-19-free) was analyzed using pyrosequencing of defined CpG islands previously identified as suitable for biological age determination. The results show a consistent biological age increase in the post-COVID-19 population, determining a DeltaAge acceleration of 10.45 ± 7.29 years (+5.25 years above the range of normality) compared with 3.68 ± 8.17 years for the COVID-19-free population (p < 0.0001). A significant telomere shortening parallels this finding in the post-COVID-19 cohort compared with COVID-19-free subjects (p < 0.0001). Additionally, ACE2 expression was decreased in post-COVID-19 patients, compared with the COVID-19-free population, while DPP-4 did not change. In light of these observations, we hypothesize that some epigenetic alterations are associated with the post-COVID-19 condition, particularly in younger patients (< 60 years).
Asunto(s)
Envejecimiento/genética , COVID-19/genética , COVID-19/fisiopatología , Islas de CpG , Acortamiento del Telómero , Telómero/metabolismo , Adulto , Anciano , Enzima Convertidora de Angiotensina 2/sangre , Biomarcadores , COVID-19/complicaciones , COVID-19/etiología , Metilación de ADN , Dipeptidil Peptidasa 4/sangre , Epigenómica , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Microbiota-Huesped , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Sobrevivientes , Síndrome Post Agudo de COVID-19RESUMEN
Hirschsprung (HSCR) Associated Enterocolitis (HAEC) is a common life-threatening complication in HSCR. HAEC is suggested to be due to a loss of gut homeostasis caused by impairment of immune system, barrier defense, and microbiome, likely related to genetic causes. No gene has been claimed to contribute to HAEC occurrence, yet. Genetic investigation of HAEC by Whole-Exome Sequencing (WES) on 24 HSCR patients affected (HAEC) or not affected (HSCR-only) by enterocolitis and replication of results on a larger panel of patients allowed the identification of the HAEC susceptibility variant p.H187Q in the Oncostatin-M receptor (OSMR) gene (14.6% in HAEC and 5.1% in HSCR-only, p = 0.0024). Proteomic analysis on the lymphoblastoid cell lines from one HAEC patient homozygote for this variant and one HAEC patient not carrying the variant revealed two well distinct clusters of proteins significantly up or downregulated upon OSM stimulation. A marked enrichment in immune response pathways (q < 0.0001) was shown in the HAEC H187 cell line, while proteins upregulated in the HAEC Q187 lymphoblasts sustained pathways likely involved in pathogen infection and inflammation. In conclusion, OSMR p.H187Q is an HAEC susceptibility variant and perturbates the downstream signaling cascade necessary for the gut immune response and homeostasis maintenance.
Asunto(s)
Susceptibilidad a Enfermedades , Enterocolitis/etiología , Enterocolitis/metabolismo , Enfermedad de Hirschsprung/complicaciones , Enfermedad de Hirschsprung/genética , Subunidad beta del Receptor de Oncostatina M/genética , Transducción de Señal , Alelos , Enterocolitis/patología , Expresión Génica , Frecuencia de los Genes , Variación Genética , Genotipo , Enfermedad de Hirschsprung/diagnóstico , Humanos , Modelos Moleculares , Subunidad beta del Receptor de Oncostatina M/química , Subunidad beta del Receptor de Oncostatina M/metabolismo , Conformación Proteica , Proteómica/métodos , Relación Estructura-Actividad , Secuenciación del Exoma , Secuenciación Completa del GenomaRESUMEN
Paired Like homeobox 2B (PHOX2B) is a gene crucial for the differentiation of the neural lineages of the autonomic nervous system (ANS), whose coding mutations cause congenital central hypoventilation syndrome (CCHS). The vast majority of PHOX2B mutations in CCHS is represented by expansions of a polyalanine region in exon 3, collectively defined PARMs (PolyAlanine Repeat Mutations), the minority being frameshift, missense and nonsense mutations, defined as NPARMs (Non-PARMs). While PARMs are nearly exclusively associated with isolated CCHS, most of NPARMs is detected in syndromic CCHS, presenting with neuroblastoma and/or Hirschsprung disease. More recently, evidence of a complex role of PHOX2B in the pathogenesis of a wider spectrum of ANS disorders has emerged. Indeed, common and hypomorphic PHOX2B variants, including synonymous, polyalanine-contractions, gene deletions may influence the occurrence of either apparent life-threatening event (ALTE), Sudden Infant Death Syndrome (SIDS), neuroblastoma, or isolated HSCR, likely through small effects on PHOX2B expression levels. After an introduction to the role of PHOX2B in the ANS development, causative mutations, common variants, and gene expression deregulation of the PHOX2B gene are discussed, though the involvement of synonymous variants and contractions requires further confirmations with respect to ANS disorders and molecular mechanisms underlying the PHOX2B phenotypic heterogeneity.
Asunto(s)
Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/genética , Hipoventilación/congénito , Apnea Central del Sueño/genética , Muerte Súbita del Lactante/genética , Factores de Transcripción/genética , Sistema Nervioso Autónomo/patología , Eliminación de Gen , Humanos , Hipoventilación/epidemiología , Hipoventilación/genética , Hipoventilación/patología , Recién Nacido , Mutación/genética , Neuroblastoma/epidemiología , Neuroblastoma/genética , Neuroblastoma/patología , Apnea Central del Sueño/epidemiología , Apnea Central del Sueño/patología , Muerte Súbita del Lactante/epidemiologíaRESUMEN
RNA epigenetics is perhaps the most recent field of interest for translational epigeneticists. RNA modifications create such an extensive network of epigenetically driven combinations whose role in physiology and pathophysiology is still far from being elucidated. Not surprisingly, some of the players determining changes in RNA structure are in common with those involved in DNA and chromatin structure regulation, while other molecules seem very specific to RNA. It is envisaged, then, that new small molecules, acting selectively on RNA epigenetic changes, will be reported soon, opening new therapeutic interventions based on the correction of the RNA epigenetic landscape. In this review, we shall summarize some aspects of RNA epigenetics limited to those in which the potential clinical translatability to cardiovascular disease is emerging.
Asunto(s)
Enfermedades Cardiovasculares , Epigénesis Genética , Conformación de Ácido Nucleico , ARN , Transducción de Señal/genética , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Humanos , ARN/genética , ARN/metabolismoRESUMEN
The WHO estimated around 41 million deaths worldwide each year for age-related non-communicable chronic diseases. Hence, developing strategies to control the accumulation of cell senescence in living organisms and the overall aging process is an urgently needed problem of social relevance. During aging, many biological processes are altered, which globally induce the dysfunction of the whole organism. Cell senescence is one of the causes of this modification. Nowadays, several drugs approved for anticancer therapy have been repurposed to treat senescence, and others are under scrutiny in vitro and in vivo to establish their senomorphic or senolytic properties. In some cases, this research led to a significant increase in cell survival or to a prolonged lifespan in animal models, at least. Senomorphics can act to interfere with a specific pathway in order to restore the appropriate cellular function, preserve viability, and to prolong the lifespan. On the other hand, senolytics induce apoptosis in senescent cells allowing the remaining non-senescent population to preserve or restore tissue function. A large number of research articles and reviews recently addressed this topic. Herein, we would like to focus attention on those chemical agents with senomorphic or senolytic properties that perspectively, according to literature, suggest a potential application as senotherapeutics for chronic diseases.
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Antineoplásicos/uso terapéutico , Enfermedad Crónica/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Senescencia Celular , Enfermedad Crónica/mortalidad , Ensayos Clínicos como Asunto , Salud Global , Humanos , Neoplasias/mortalidad , Transducción de Señal/efectos de los fármacosRESUMEN
Heterozygous mutations in the PHOX2B gene are causative of congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by defective autonomic control of breathing due to the impaired differentiation of neural crest cells. Among PHOX2B mutations, polyalanine (polyAla) expansions are almost exclusively associated with isolated CCHS, whereas frameshift variants, although less frequent, are often more severe than polyAla expansions and identified in syndromic CCHS. This article provides a complete review of all the frameshift mutations identified in cases of isolated and syndromic CCHS reported in the literature as well as those identified by us and not yet published. These were considered in terms of both their structure, whether the underlying indels induced frameshifts of either 1 or 2 steps ("frame 2" and "frame 3" mutations respectively), and clinical associations. Furthermore, we evaluated the structural and functional effects of one "frame 3" mutation identified in a patient with isolated CCHS, and one "frame 2" mutation identified in a patient with syndromic CCHS, also affected with Hirschsprung's disease and neuroblastoma. The data thus obtained confirm that the type of translational frame affects the severity of the transcriptional dysfunction and the predisposition to isolated or syndromic CCHS.
Asunto(s)
Mutación del Sistema de Lectura/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Hipoventilación/congénito , Apnea Central del Sueño/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Humanos , Hipoventilación/genética , Hipoventilación/metabolismo , Microscopía Fluorescente , Mutación , Apnea Central del Sueño/metabolismoRESUMEN
Curcumin has been reported to inhibit inflammation, tumor growth, angiogenesis and metastasis by decreasing cell growth and by inducing apoptosis mainly through the inhibition of nuclear factor kappa-B (NFκB), a master regulator of inflammation. Recent reports also indicate potential metabolic effects of the polyphenol, therefore we analyzed whether and how it affects the energy metabolism of tumor cells. We show that curcumin (10 µM) inhibits the activity of ATP synthase in isolated mitochondrial membranes leading to a dramatic drop of ATP and a reduction of oxygen consumption in in vitro and in vivo tumor models. The effects of curcumin on ATP synthase are independent of the inhibition of NFκB since the IκB Kinase inhibitor, SC-514, does not affect ATP synthase. The activities of the glycolytic enzymes hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase are only slightly affected in a cell type-specific manner. The energy impairment translates into decreased tumor cell viability. Moreover, curcumin induces apoptosis by promoting the generation of reactive oxygen species (ROS) and malondialdehyde (MDA), a marker of lipid oxidation, and autophagy, at least in part due to the activation of the AMP-activated protein kinase (AMPK). According to the in vitro anti-tumor effect, curcumin (30 mg/kg body weight) significantly delayed in vivo cancer growth likely due to an energy impairment but also through the reduction of tumor angiogenesis. These results establish the ATP synthase, a central enzyme of the cellular energy metabolism, as a target of the antitumoral polyphenol leading to inhibition of cancer cell growth and a general reprogramming of tumor metabolism.
Asunto(s)
Antineoplásicos/uso terapéutico , Curcumina/uso terapéutico , Metabolismo Energético/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Consumo de Oxígeno/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Hexoquinasa/metabolismo , Quinasa I-kappa B/antagonistas & inhibidores , L-Lactato Deshidrogenasa/metabolismo , Malondialdehído/metabolismo , Ratones , Ratones Desnudos , Neovascularización Patológica/tratamiento farmacológico , Fosfofructoquinasas/metabolismo , Piruvato Quinasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tiofenos/farmacologíaRESUMEN
Chromogranin A (CgA), a secretory protein released in the blood by neuroendocrine cells and neurons, is the precursor of various bioactive fragments involved in the regulation of the cardiovascular system, metabolism, innate immunity, angiogenesis, and tissue repair. After the original demonstration that circulating CgA can serve as a biomarker for a wide range of neuroendocrine tumors, several studies have shown that increased levels of CgA can be present also in the blood of patients with cardiovascular, gastrointestinal, and inflammatory diseases with, in certain cases, important diagnostic and prognostic implications. Considering the high structural and functional heterogeneity of the CgA system, comprising precursor and fragments, it is not surprising that the different immunoassays used in these studies led, in some cases, to discrepant results. Here, we review these notions and we discuss the importance of measuring total-CgA, full-length CgA, specific fragments, and their relative levels for a more thorough assessment of the pathophysiological function and diagnostic/prognostic value of the CgA system.
Asunto(s)
Biomarcadores de Tumor/sangre , Cromogranina A/sangre , Tumores Neuroendocrinos/sangre , Animales , Cromogranina A/química , Humanos , Fragmentos de Péptidos/sangreRESUMEN
HSCR is a congenital disorder of the enteric nervous system, characterized by the absence of neurons along a variable length of the gut resulting from loss-of-function RET mutations. Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by impaired response to hypercapnia and hypoxemia caused by heterozygous mutations of the PHOX2B gene, mostly polyalanine (polyA) expansions but also missense, nonsense, and frameshift mutations, while polyA contractions are common in the population and believed neutral. HSCR associated CCHS can present in patients carrying PHOX2B mutations. Indeed, RET expression is orchestrated by different transcriptional factors among which PHOX2B, thus suggesting its possible role in HSCR pathogenesis. Following the observation of HSCR patients carrying in frame trinucleotide deletions within the polyalanine stretch in exon 3 (polyA contractions), we have verified the hypothesis that these PHOX2B variants do reduce its transcriptional activity, likely resulting in a down-regulation of RET expression and, consequently, favouring the development of the HSCR phenotype. Using proper reporter constructs, we show here that the in vitro transactivation of the RET promoter by different HSCR-associated PHOX2B polyA variants has resulted significantly lower compared to the effect of PHOX2B wild type protein. In particular, polyA contractions do induce a reduced transactivation of the RET promoter, milder compared to the severe polyA expansions associated with CCHS+HSCR, and correlated with the length of the deleted trait, with a more pronounced effect when contractions are larger.
Asunto(s)
Secuencia de Bases , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Enfermedad de Hirschsprung/metabolismo , Proteínas de Homeodominio/metabolismo , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-ret/biosíntesis , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Transcripción Genética , Línea Celular Tumoral , Femenino , Enfermedad de Hirschsprung/genética , Enfermedad de Hirschsprung/patología , Proteínas de Homeodominio/genética , Humanos , Masculino , Péptidos/genética , Proteínas Proto-Oncogénicas c-ret/genética , Factores de Transcripción/genéticaRESUMEN
Neuroblastoma (NB) is a rare childhood cancer of the peripheral sympathetic nervous system and accounts for approximately 10% of all pediatric tumors. Heterozygous PHOX2B mutations have been found in association with NB development in familial, sporadic and syndromic cases. In addition, the PHOX2B gene is widely over-expressed both in tumor samples and NB cell lines. Post-transcriptional gene regulation is known to be involved in mRNA stability and, in NB, microRNAs (miRNAs) seem to be responsible for altered expression of genes driving differentiation, apoptosis, and migration. To assess the possible impact of post-transcriptional regulation in NB cell lines, we have focused on the PHOX2B mRNA stability by both in silico analysis and functional studies on its 3'untranslated region (3'UTR). PHOX2B gene expression has resulted under post-transcriptional control, as suggested by: i) instability of PHOX2B mRNA, demonstrated by short mRNA half-life levels in both IMR32 and LAN-1 cell lines, ii) role of the PHOX2B-3'UTR, confirmed by the activity of proper reporter constructs, and iii) miRNA-204, shown to enhance the PHOX2B 3'UTR mediated down-regulation of the reporter construct activity. Finally, miRNA-204 has resulted to decrease the stability of the PHOX2B mRNA at different extents in the presence of different SNP rs1063611 alleles. Therefore, post-transcriptional down-regulation of the PHOX2B gene takes place in NB cell lines and miRNA-204 participates in such a 3'UTR mediated control.
Asunto(s)
Neoplasias Encefálicas/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , MicroARNs/fisiología , Neuroblastoma/genética , Factores de Transcripción/genética , Regiones no Traducidas 3' , Secuencia de Bases , Neoplasias Encefálicas/patología , Células Cultivadas , Niño , Regulación hacia Abajo/genética , Humanos , Datos de Secuencia Molecular , Neuroblastoma/patología , Procesamiento Postranscripcional del ARN/genéticaRESUMEN
PHOX2B is a transcription factor involved in the regulation of neurogenesis and in the correct differentiation of the autonomic nervous system. The pathogenetic role of PHOX2B in neuroblastoma (NB) is supported by mutations in familial, sporadic and syndromic cases of NB and overexpression of PHOX2B and its target ALK in tumor samples and NB cell lines. Starting from these observations, we have performed in vitro drug screening approaches targeting PHOX2B overexpression as a potential pharmacological means in NB. In particular, in order to identify molecules able to decrease PHOX2B expression, we have evaluated the effects of 70 compounds in IMR-32 cell line stably expressing the luciferase gene under the control of the PHOX2B promoter. Curcumin, SAHA and trichostatin A showed to down-regulate the PHOX2B promoter activity which resulted in a decrease of both protein and mRNA expressions. In addition, we have observed that curcumin acts by interfering with PBX-1/MEIS-1, NF-κB and AP-1 complexes, in this work demonstrated for the first time to regulate the transcription of the PHOX2B gene. Finally, combined drug treatments showed successful effects in down-regulating the expression of both PHOX2B and its target ALK genes, thus supporting the notion of the effectiveness of molecule combination in tumor therapy.
Asunto(s)
Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Proteínas de Homeodominio/antagonistas & inhibidores , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Western Blotting , Diferenciación Celular , Curcumina/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Técnicas In Vitro , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neuroblastoma/patología , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
The PHOX2B transcription factor plays a crucial role in autonomic nervous system development. In humans, heterozygous mutations of the PHOX2B gene lead to congenital central hypoventilation syndrome (CCHS), a rare disorder characterized by a broad variety of symptoms of autonomic nervous system dysfunction including inadequate control of breathing. The vast majority of patients with CCHS are heterozygous for a polyalanine repeat expansion mutation involving a polyalanine tract of twenty residues in the C-terminus of PHOX2B. Although several lines of evidence support a dominant-negative mechanism for PHOX2B mutations in CCHS, the molecular effects of PHOX2B mutant proteins on the transcriptional activity of the wild-type protein have not yet been elucidated. As one of the targets of PHOX2B is the PHOX2B gene itself, we tested the transcriptional activity of wild-type and mutant proteins on the PHOX2B gene promoter, and found that the transactivation ability of proteins with polyalanine expansions decreased as a function of the length of the expansion, whereas DNA binding was severely affected only in the case of the mutant with the longest polyalanine tract (+13 alanine). Co-transfection experiments using equimolar amounts of PHOX2B wild-type and mutant proteins in order to simulate a heterozygous state in vitro and four different PHOX2B target gene regulatory regions (PHOX2B, PHOX2A, DBH, TLX2) clearly showed that the polyalanine expanded proteins alter the transcriptional activity of wild-type protein in a promoter-specific manner, without any clear correlation with the length of the expansion. Moreover, although reduced transactivation may be caused by retention of the wild-type protein in the cytoplasm or in nuclear aggregates, this mechanism can only be partially responsible for the pathogenesis of CCHS because of the reduction in cytoplasmic and nuclear accumulation when the +13 alanine mutant is co-expressed with wild-type protein, and the fact that the shortest polyalanine expansions do not form visible cytoplasmic aggregates. Deletion of the C-terminal of PHOX2B leads to a protein that correctly localizes in the nucleus but impairs PHOX2B wild-type transcriptional activity, thus suggesting that protein mislocalization is not the only mechanism leading to CCHS. The results of this study provide novel in vitro experimental evidence of a transcriptional dominant-negative effect of PHOX2B polyalanine mutant proteins on wild-type protein on two different PHOX2B target genes.
Asunto(s)
Expansión de las Repeticiones de ADN/genética , Proteínas de Homeodominio/genética , Hipoventilación/congénito , Apnea Central del Sueño/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Western Blotting , Ensayo de Cambio de Movilidad Electroforética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Hipoventilación/genética , Mutación , Péptidos , Regiones Promotoras Genéticas/genética , Transcripción Genética , TransfecciónRESUMEN
OBJECTIVES: Tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is caused by TNFRSF1A mutations, known to induce intracellular retention of the TNFα receptor 1 (TNFR1) protein, defective TNFα-induced apoptosis, and production of reactive oxygen species. As downregulation of autophagy, the main cellular pathway involved in insoluble aggregate elimination, has been observed to increase the inflammatory response, we investigated whether it plays a role in TRAPS pathogenesis. METHODS: The possible link between TNFRSF1A mutations and inflammation in TRAPS was studied in HEK-293T cells, transfected with expression constructs for wild-type and mutant TNFR1 proteins, and in monocytes derived from patients with TRAPS, by investigating autophagy function, NF-κB activation and interleukin (IL)-1ß secretion. RESULTS: We found that autophagy is responsible for clearance of wild-type TNFR1, but when TNFR1 is mutated, the autophagy process is defective, probably accounting for mutant TNFR1 accumulation as well as TRAPS-associated induction of NF-κB activity and excessive IL-1ß secretion, leading to chronic inflammation. Autophagy inhibition due to TNFR1 mutant proteins can be reversed, as demonstrated by the effects of the antibiotic geldanamycin, which was found to rescue the membrane localisation of mutant TNFR1 proteins, reduce their accumulation and counteract the increased inflammation by decreasing IL-1ß secretion. CONCLUSIONS: Autophagy appears to be an important mechanism in the pathogenesis of TRAPS, an observation that provides a rationale for the most effective therapy in this autoinflammatory disorder. Our findings also suggest that autophagy could be proposed as a novel therapeutic target for TRAPS and possibly other similar diseases.
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Autofagia/genética , Enfermedades Autoinflamatorias Hereditarias/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Adolescente , Adulto , Autofagia/fisiología , Estudios de Casos y Controles , Niño , Preescolar , Células HEK293 , Humanos , Interleucina-1beta/metabolismo , Persona de Mediana Edad , FN-kappa B/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , SíndromeRESUMEN
Heterozygous mutations of the GFAP gene are responsible for Alexander disease, a neurodegenerative disorder characterized by intracytoplasmic Rosenthal fibers (RFs) in dystrophic astrocytes. In vivo and in vitro models have shown co-localization of mutant GFAP proteins with the small heat shock proteins (sHSPs) HSP27 and alphaB-crystallin, ubiquitin and proteasome components. Results reported by several recent studies agree on ascribing an altered cytoskeletal pattern to mutant GFAP proteins, an effect which induces mutant proteins accumulation, leading to impaired proteasome function and autophagy induction. On the basis of the protective role shown by both these small heat shock proteins (sHSPs), and on the already well established neuroprotective effects of curcumin in several diseases, we have investigated the effects of this compound in an in vitro model of Alexander disease, consisting in U251-MG astrocytoma cells transiently transfected with a construct encoding for GFAP carrying the p.R239C mutation in frame with the reporter green fluorescent protein (GFP). In particular, depending on the dose used, we have observed that curcumin is able to induce both HSP27 and alphaB-crystallin, to reduce expression of both RNA and protein of endogenous GFAP, to induce autophagy and, finally, to rescue the filamentous organization of the GFAP mutant protein, thus suggesting a role of this spice in counteracting the pathogenic effects of GFAP mutations.
Asunto(s)
Enfermedad de Alexander/tratamiento farmacológico , Enfermedad de Alexander/metabolismo , Curcumina/farmacología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Enfermedad de Alexander/genética , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Regulación hacia Abajo/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Multimerización de Proteína/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Background: During the COVID-19 pandemic, telemedicine has been recognised as a powerful modality to shorten the length of hospital stay and to free up beds for the sicker patients. Lombardy, and in particular the areas of Bergamo, Brescia, and Milan, was one of the regions in Europe most hit by the COVID-19 pandemic. The primary aim of the MIRATO project was to compare the incidence of severe events (hospital readmissions and mortality) in the first three months after discharge between COVID-19 patients followed by a Home-Based Teleassistance and Teleconsultation (HBTT group) program and those discharged home without Telemedicine support (non-HBTT group). Methods: The study was designed as a matched case-control study. The non-HBTT patients were matched with the HBTT patients for sex, age, presence of COVID-19 pneumonia and number of comorbidities. After discharge, the HBTT group underwent a telecare nursing and specialist teleconsultation program at home for three months, including monitoring of vital signs and symptoms. Further, in this group we analysed clinical data, patients' satisfaction with the program, and quality of life. Results: Four hundred twenty-two patients per group were identified for comparison. The median age in both groups was 70 ± 11 years (62% males). One or more comorbidities were present in 86% of the HBTT patients and 89% in the non-HBTT group (p = ns). The total number of severe events was 17 (14 hospitalizations and 3 deaths) in the HBTT group and 40 (26 hospitalizations and 16 deaths) in the non-HBTT group (p = 0.0007). The risk of hospital readmission or death after hospital discharge was significantly lower in HBTT patients (Log-rank Test p = 0.0002). In the HBTT group, during the 3-month follow-up, 5,355 teleassistance contacts (13 ± 4 per patient) were performed. The number of patients with one or more symptoms declined significantly: from 338 (78%) to 183 (45%) (p < 0.00001). Both the physical (ΔPCS12: 5.9 ± 11.4) component and the mental (ΔMCS12: 4.4 ± 12.7) component of SF-12 improved significantly (p < 0.0001). Patient satisfaction with the program was very high in all participants. Conclusions: Compared to usual care, an HBTT program can reduce severe events (hospital admissions/mortality) at 3-months from discharge and improve symptoms and quality of life. Clinical trial registration: www.ClinicalTrials.gov, NCT04898179.
RESUMEN
Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1. Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition. Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness. Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood-brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases "in one shot." Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment.