RESUMEN

Angelman syndrome is a severe neurodevelopmental disorder secondary to disruption of the UBE3A gene in the maternal allele of chromosome 15. Its manifestations are mainly neurological, but a multidisciplinary management is required for its treatment. There are consensus guidelines available for best clinical management. Current clinical trials with antisense oligonucleotides promise, for the first time, to treat the cause by activating the UBE3A gene in the paternal allele, showing encouraging preliminary clinical effects. Inoculation of UBE3A gene through a viral vector has been tested in animal models and is underway for future clinical trials.

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El síndrome de Angelman es un grave desorden del neurodesarrollo secundario a disrupción del gen UBE3A en el alelo materno del cromosoma 15. Sus manifestaciones son principalmente neurológicas, pero se requiere de un manejo multidisciplinario para su tratamiento. Existen guías por consenso para el manejo clínico adecuado. Actuales ensayos clínicos con oligonucleótidos antisentido prometen, por primera vez, tratar la causa por medio de activación del gen UBE3A en el alelo paterno, demostrando efectos clínicos preliminares alentadores. La inoculación del gen UBE3A a través de un vector viral ha sido probada en modelos animales y está en vías para futuros ensayos clínicos.

Asunto(s)

Síndrome de Angelman , Terapia Genética , Humanos , Síndrome de Angelman/terapia , Síndrome de Angelman/genética , Oligonucleótidos Antisentido/uso terapéutico , Ubiquitina-Proteína Ligasas/genética , Animales

RESUMEN

Mammalian Ste-20-like Kinases 1 and 2 (MST1/2) are core serine-threonine kinases of the Hippo pathway regulating several cellular processes, including cell cycle arrest and cell death. Here, we discovered a novel alternative splicing variant of the MST2 encoding gene, STK3, in malignant cells and tumor datasets. This variant, named STK3∆7 or MST2∆7 (for mRNA or protein, respectively), resulted from the skipping of exon 7. MST2∆7 exhibited increased ubiquitylation and interaction with the E3 ubiquitin-protein ligase CHIP compared to the full-length protein (MST2FL). Exon 7 in STK3 encodes a segment within the kinase domain, and its exclusion compromised MST2 interaction with and phosphorylation of MOB, a major MST1/2 substrate. Nevertheless, MST2∆7 was capable of interacting with MST1 and MST2FL. Unlike MST2FL, overexpression of MST2∆7 did not lead to increased cell death and growth arrest. Strikingly, we observed the exclusion of STK3 exon 7 in 3.2-15% of tumor samples from patients of several types of cancer, while STK3∆7 was seldomly found in healthy tissues. Our study identified a novel STK3 splicing variant with loss of function and the potential to disturb tissue homeostasis by impacting on MST2 activities in the regulation of cell death and quiescence.

Asunto(s)

Empalme Alternativo , Proliferación Celular , Proteínas Serina-Treonina Quinasas , Serina-Treonina Quinasa 3 , Humanos , Proteínas Adaptadoras Transductoras de Señales , Línea Celular Tumoral , Exones/genética , Células HEK293 , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Serina-Treonina Quinasa 3/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/genética

RESUMEN

Chikungunya (CHIKV) and Mayaro (MAYV) viruses are arthritogenic alphaviruses that promote an incapacitating and long-lasting inflammatory muscle-articular disease. Despite studies pointing out the importance of skeletal muscle (SkM) in viral pathogenesis, the long-term consequences on its physiology and the mechanism of persistence of symptoms are still poorly understood. Combining molecular, morphological, nuclear magnetic resonance imaging, and histological analysis, we conduct a temporal investigation of CHIKV and MAYV replication in a wild-type mice model, focusing on the impact on SkM composition, structure, and repair in the acute and late phases of infection. We found that viral replication and induced inflammation promote a rapid loss of muscle mass and reduction in fiber cross-sectional area by upregulation of muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1 expression, both key regulators of SkM fibers atrophy. Despite a reduction in inflammation and clearance of infectious viral particles, SkM atrophy persists until 30 days post-infection. The genomic CHIKV and MAYV RNAs were still detected in SkM in the late phase, along with the upregulation of chemokines and anti-inflammatory cytokine expression. In agreement with the involvement of inflammatory mediators on induced atrophy, the neutralization of TNF and a reduction in oxidative stress using monomethyl fumarate, an agonist of Nrf2, decreases atrogen expression and atrophic fibers while increasing weight gain in treated mice. These data indicate that arthritogenic alphavirus infection could chronically impact body SkM composition and also harm repair machinery, contributing to a better understanding of mechanisms of arthritogenic alphavirus pathogenesis and with a description of potentially new targets of therapeutic intervention.

Asunto(s)

Virus Chikungunya , Músculo Esquelético , Atrofia Muscular , Estrés Oxidativo , Animales , Atrofia Muscular/virología , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Ratones , Músculo Esquelético/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/virología , Fiebre Chikungunya/patología , Fiebre Chikungunya/virología , Fiebre Chikungunya/metabolismo , Inflamación/patología , Inflamación/metabolismo , Inflamación/virología , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Replicación Viral , Ratones Endogámicos C57BL , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Infecciones por Alphavirus/virología , Infecciones por Alphavirus/patología , Infecciones por Alphavirus/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Modelos Animales de Enfermedad , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética

RESUMEN

Cullin-1-RING ubiquitin ligases (CRL1) or SCF1 (SKP1-CUL1-RBX1) E3 ubiquitin ligases are the largest and most extensively investigated class of E3 ligases in mammals that regulate fundamental processes, such as the cell cycle and proliferation. These enzymes are multiprotein complexes comprising SKP1, CUL1, RBX1, and an F-box protein that acts as a specificity factor by interacting with SKP1 through its F-box domain and recruiting substrates via other domains. E3 ligases are important players in the ubiquitination process, recognizing and transferring ubiquitin to substrates destined for degradation by proteasomes or processing by deubiquitinating enzymes. The ubiquitin-proteasome system (UPS) is the main regulator of intracellular proteolysis in eukaryotes and is required for parasites to alternate hosts in their life cycles, resulting in successful parasitism. Leishmania UPS is poorly investigated, and CRL1 in L. infantum, the causative agent of visceral leishmaniasis in Latin America, is yet to be described. Here, we show that the L. infantum genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein-like protein), and LINF_210005300 (ring-box protein 1 -putative) form a LinfCRL1 complex structurally similar to the H. sapiens CRL1. Mass spectrometry analysis of the LinfSkp1 and LinfCul1 interactomes revealed proteins involved in several intracellular processes, including six F-box proteins known as F-box-like proteins (Flp) (data are available via ProteomeXchange with identifier PXD051961). The interaction of LinfFlp 1-6 with LinfSkp1 was confirmed, and using in vitro ubiquitination assays, we demonstrated the function of the LinfCRL1(Flp1) complex to transfer ubiquitin. We also found that LinfSKP1 and LinfRBX1 knockouts resulted in nonviable L. infantum lineages, whereas LinfCUL1 was involved in parasite growth and rosette formation. Finally, our results suggest that LinfCul1 regulates the S phase progression and possibly the transition between the late S to G2 phase in L. infantum. Thus, a new class of E3 ubiquitin ligases has been described in L. infantum with functions related to various parasitic processes that may serve as prospective targets for leishmaniasis treatment.

Asunto(s)

Proteínas Cullin , Leishmania infantum , Leishmania infantum/metabolismo , Leishmania infantum/enzimología , Proteínas Cullin/metabolismo , Proteínas Cullin/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Ubiquitinación , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/metabolismo , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo

RESUMEN

OBJECTIVES: The etiology of central precocious puberty (CPP) has expanded with identification of new genetic causes, including the monogenic deficiency of Makorin-Ring-Finger-Protein-3 (MKRN3). We aimed to assess the prevalence of CPP causes and the predictors of genetic involvement in this phenotype. DESIGN: A retrospective cohort study for an etiological survey of patients with CPP from a single academic center. METHODS: All patients with CPP had detailed medical history, phenotyping, and brain magnetic resonance imaging (MRI); those with negative brain MRI (apparently idiopathic) were submitted to genetic studies, mainly DNA sequencing studies, genomic microarray, and methylation analysis. RESULTS: We assessed 270 patients with CPP: 50 (18.5%) had CPP-related brain lesions (34 [68%] congenital lesions), whereas 220 had negative brain MRI. Of the latter, 174 (165 girls) were included for genetic studies. Genetic etiologies were identified in 22 patients (20 girls), indicating an overall frequency of genetic CPP of 12.6% (22.2% in boys and 12.1% in girls). The most common genetic defects were MKRN3, Delta-Like-Non-Canonical-Notch-Ligand-1 (DLK1), and Methyl-CpG-Binding-Protein-2 (MECP2) loss-of-function mutations, followed by 14q32.2 defects (Temple syndrome). Univariate logistic regression identified family history (odds ratio [OR] 3.3; 95% CI 1.3-8.3; P = .01) and neurodevelopmental disorders (OR 4.1; 95% CI 1.3-13.5; P = .02) as potential clinical predictors of genetic CPP. CONCLUSIONS: Distinct genetic causes were identified in 12.6% patients with apparently idiopathic CPP, revealing the genetic etiology as a relevant cause of CPP in both sexes. Family history and neurodevelopmental disorders were suggested as predictors of genetic CPP. We originally proposed an algorithm to investigate the etiology of CPP including genetic studies.

Asunto(s)

Pubertad Precoz , Humanos , Pubertad Precoz/genética , Pubertad Precoz/etiología , Pubertad Precoz/epidemiología , Femenino , Masculino , Niño , Estudios Retrospectivos , Preescolar , Imagen por Resonancia Magnética , Ribonucleoproteínas/genética , Estudios de Cohortes , Ubiquitina-Proteína Ligasas/genética , Mutación , Encéfalo/diagnóstico por imagen

RESUMEN

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease globally, with a fast-growing prevalence. The etiology of PD exhibits a multifactorial complex nature and remains challenging. Herein, we described clinical, molecular, and integrative bioinformatics findings from a Brazilian female affected by Early-Onset PD (EOPD) harboring a recurrent homozygous pathogenic deletion in the parkin RBR E3 ubiquitin protein ligase gene (PRKN; NM_004562.3:c.155delA; p.Asn52Metfs*29; rs754809877), along with a novel heterozygous variant in the synaptojanin 1 gene (SYNJ1; NM_003895.3:c.62G > T; p.Cys21Phe; rs1486511197) found by Whole Exome Sequencing. Uncommon or unreported PRKN-related clinical features in the patient include cognitive decline, auditory and visual hallucinations, REM sleep disorder, and depression, previously observed in SYNJ1-related conditions. Moreover, PRKN interacts with endophilin A1, which is a major binding partner of SYNJ1. This protein plays a pivotal role in regulating the dynamics of synaptic vesicles, particularly in the context of endocytosis and recycling processes. Altogether, our comprehensive analyses underscore a potential synergistic effect between the PRKN and SYNJ1 variants over the pathogenesis of EOPD.

Asunto(s)

Enfermedad de Parkinson , Ubiquitina-Proteína Ligasas , Humanos , Enfermedad de Parkinson/genética , Femenino , Ubiquitina-Proteína Ligasas/genética , Adulto , Edad de Inicio , Proteínas del Tejido Nervioso/genética , Monoéster Fosfórico Hidrolasas

RESUMEN

OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.

Asunto(s)

Neoplasias de la Mama , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Metilación de ADN , Proteínas de Unión a Retinoblastoma , Adulto , Anciano , Femenino , Humanos , Persona de Mediana Edad , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/genética , Ácidos Nucleicos Libres de Células/genética , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/análisis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN/genética , Mastectomía , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Proteínas de Unión a Retinoblastoma/genética , Ubiquitina-Proteína Ligasas/genética

RESUMEN

BACKGROUND: The activated microglia have been reported as pillar factors in neuropathic pain (NP) pathology, but the molecules driving pain-inducible microglial activation require further exploration. In this study, we investigated the effect of dorsal root ganglion (DRG)-derived exosomes (Exo) on microglial activation and the related mechanism. METHODS: A mouse model of NP was generated by spinal nerve ligation (SNL), and DRG-derived Exo were extracted. The effects of DRG-Exo on NP and microglial activation in SNL mice were evaluated using behavioral tests, HE staining, immunofluorescence, and western blot. Next, the differentially enriched microRNAs (miRNAs) in DRG-Exo-treated microglia were analyzed using microarrays. RT-qPCR, RNA pull-down, dual-luciferase reporter assay, and immunofluorescence were conducted to verify the binding relation between miR-16-5p and HECTD1. Finally, the effects of ubiquitination modification of HSP90 by HECTD1 on NP progression and microglial activation were investigated by Co-IP, western blot, immunofluorescence assays, and rescue experiments. RESULTS: DRG-Exo aggravated NP resulting from SNL in mice, promoted the activation of microglia in DRG, and increased neuroinflammation. miR-16-5p knockdown in DRG-Exo alleviated the stimulating effects of DRG-Exo on NP and microglial activation. DRG-Exo regulated the ubiquitination of HSP90 through the interaction between miR-16-5p and HECTD1. Ubiquitination alteration of HSP90 was involved in microglial activation during NP. CONCLUSIONS: miR-16-5p shuttled by DRG-Exo regulated the ubiquitination of HSP90 by interacting with HECTD1, thereby contributing to the microglial activation in NP.

Asunto(s)

Exosomas , Ganglios Espinales , Proteínas HSP90 de Choque Térmico , MicroARNs , Microglía , Neuralgia , Animales , Masculino , Ratones , Modelos Animales de Enfermedad , Exosomas/metabolismo , Ganglios Espinales/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Ratones Endogámicos C57BL , Microglía/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Neuralgia/metabolismo , Neuralgia/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética

RESUMEN

Shade avoidance syndrome is an important adaptive strategy. Under shade, major transcriptional rearrangements underlie the reallocation of resources to elongate vegetative structures and redefine the plant architecture to compete for photosynthesis. BBX28 is a B-box transcription factor involved in seedling de-etiolation and flowering in Arabidopsis (Arabidopsis thaliana), but its function in shade-avoidance response is completely unknown. Here, we studied the function of BBX28 using two mutant and two transgenic lines of Arabidopsis exposed to white light and simulated shade conditions. We found that BBX28 promotes hypocotyl growth under shade through the phytochrome system by perceiving the reduction of red photons but not the reduction of photosynthetically active radiation or blue photons. We demonstrated that hypocotyl growth under shade is sustained by the protein accumulation of BBX28 in the nuclei in a CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1)-dependent manner at the end of the photoperiod. BBX28 up-regulates the expression of transcription factor- and auxin-related genes, thereby promoting hypocotyl growth under prolonged shade. Overall, our results suggest the role of BBX28 in COP1 signaling to sustain the shade-avoidance response and extend the well-known participation of other members of BBX transcription factors for fine-tuning plant growth under shade.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Hipocótilo , Luz , Factores de Transcripción , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Hipocótilo/crecimiento & desarrollo , Hipocótilo/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Plantas Modificadas Genéticamente , Mutación/genética , Ácidos Indolacéticos/metabolismo , Fotoperiodo , Transducción de Señal/genética

RESUMEN

In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation.

Asunto(s)

Leishmania mexicana , Proteínas Protozoarias , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Leishmania mexicana/genética , Leishmania mexicana/enzimología , Leishmania mexicana/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Inmunoprecipitación

RESUMEN

AIM: This study aimed to investigate the effects of caffeine on pathways associated with mitochondrial quality control and mitochondrial capacity during skeletal muscle regeneration, focusing on the role of Parkin, a key protein involved in mitophagy. METHODS: We used in vitro C2C12 myoblast during differentiation with and without caffeine in the medium, and we evaluated several markers of mitochondrial quality control pathways and myotube growth. In vivo experiments, we used C57BL/6J (WT) and Parkintm 1Shn lineage (Parkin-/- ) mice and injured tibial anterior muscle. The mice regenerated TA muscle for 3, 10, and 21 days with or without caffeine ingestion. TA muscle was used to analyze the protein content of several markers of mitochondrial quality pathways, muscle satellite cell differentiation, and protein synthesis. Furthermore, it analyzed mtDNA, mitochondrial respiration, and myofiber growth. RESULTS: C2C12 differentiation experiments showed that caffeine decreased Parkin content, potentially leading to increased DRP1 and PGC-1α content and altered mitochondrial population, thereby enhancing growth capacity. Using Parkin-/- mice, we found that caffeine intake during the regenerative process induces an increase in AMPKα phosphorylation and PGC-1α and TFAM content, changes that were partly Parkin-dependent. In addition, the absence of Parkin potentiates the ergogenic effect of caffeine by increasing mitochondrial capacity and myotube growth. Those effects are related to increased ATF4 content and activation of protein synthesis pathways, such as increased 4E-BP1 phosphorylation. CONCLUSION: These findings demonstrate that caffeine ingestion changes mitochondrial quality control during skeletal muscle regeneration, and Parkin is a central player in those mechanisms.

Asunto(s)

Cafeína , Músculo Esquelético , Ratones , Animales , Cafeína/farmacología , Músculo Esquelético/metabolismo , Ratones Endogámicos C57BL , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Regeneración

RESUMEN

PURPOSE: We investigated the impact of anthracycline-based chemotherapy on methylation status of RB1 gene in peripheral blood leukocytes together with parameters of oxidative stress and inflammation in sarcoma patients. PATIENTS/METHODS: Blood samples were collected from 51 consecutive newly diagnosed sarcoma patients admitted to University Hospital Center Zagreb (Zagreb, Croatia) for first-line chemotherapy before the first cycle and post-chemotherapy. Methylation and copy number variation (CNV) of leukocyte RB1 gene were assessed using MS-MLPA probes. In addition, in blood samples, parameters of oxidative stress (ROS, MDA, SOD, and GSH) and inflammation (CRP, WBC, and NBC) were followed. RESULTS: In pre-chemotherapy samples, no CNVs and aberrant methylation of CpG106 promoter region of RB1 gene were detected; however, one patient had hypermethylation (by approximately 10%) of imprinted locus CpG85 in intron 2 of RB1 gene. In addition, a very good correlation of the tumor burden and CRP and tumor burden and GSH was found. The anthracycline-based chemotherapy reverts methylation of RB1 gene-imprinted locus CpG85 to normal level. Moreover, inflammation and oxidative stress parameters such as CRP, WBC, ROS, and MDA were significantly decreased in post-chemotherapy samples. CONCLUSION: This single-centered study on a cohort of consecutive sarcoma patients indicates that sarcoma patients can have aberrant germline DNA methylation and confirms the relationship of tumor burden with inflammation and oxidative stress. The applied chemotherapy protocols reverted RB1 gene methylation to normal level and decreased the level of inflammation and oxidative damage, thus indicating chemotherapy benefit to the patient's health status.

Asunto(s)

Antraciclinas , Metilación de ADN , Inflamación , Leucocitos , Estrés Oxidativo , Proteínas de Unión a Retinoblastoma , Sarcoma , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Antraciclinas/uso terapéutico , Variaciones en el Número de Copia de ADN , Inflamación/genética , Leucocitos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteínas de Unión a Retinoblastoma/efectos de los fármacos , Proteínas de Unión a Retinoblastoma/genética , Sarcoma/tratamiento farmacológico , Sarcoma/genética , Sarcoma/patología , Ubiquitina-Proteína Ligasas/genética

RESUMEN

Studies in humans and mice support a role for Makorin RING finger protein 3 (MKRN3) as an inhibitor of gonadotropin-releasing hormone (GnRH) secretion prepubertally, and its loss of function is the most common genetic cause of central precocious puberty in humans. Studies have shown that the gonads can synthesize neuropeptides and express MKRN3/Mkrn3 mRNA. Therefore, we aimed to investigate the spatiotemporal expression pattern of Mkrn3 in gonads during sexual development, and its potential regulation in the functional testicular compartments by gonadotropins. Mkrn3 mRNA was detected in testes and ovaries of wild-type mice at all ages evaluated, with a sexually dimorphic expression pattern between male and female gonads. Mkrn3 expression was highest peripubertally in the testes, whereas it was lower peripubertally than prepubertally in the ovaries. Mkrn3 is expressed primarily in the interstitial compartment of the testes but was also detected at low levels in the seminiferous tubules. In vitro studies demonstrated that Mkrn3 mRNA levels increased in human chorionic gonadotropin (hCG)-treated Leydig cell primary cultures. Acute administration of a GnRH agonist in adult mice increased Mkrn3 expression in testes, whereas inhibition of the hypothalamic-pituitary-gonadal axis by chronic administration of GnRH agonist had the opposite effect. Finally, we found that hCG increased Mkrn3 mRNA levels in a dose-dependent manner. Taken together, our developmental expression analyses, in vitro and in vivo studies show that Mkrn3 is expressed in the testes, predominantly in the interstitial compartment, and that Mkrn3 expression increases after puberty and is responsive to luteinizing hormone/hCG stimulation.

Asunto(s)

Gonadotropina Coriónica , Hormona Luteinizante , Pubertad Precoz , Ubiquitina-Proteína Ligasas , Animales , Femenino , Humanos , Masculino , Ratones , Hormona Liberadora de Gonadotropina , ARN Mensajero , Ubiquitina-Proteína Ligasas/genética

RESUMEN

OBJECTIVE: Local recurrence, distant metastasis, and perineural invasion (PNI) viciously occur in salivary adenoid cystic carcinoma (SACC), resulting in a poor prognosis. This study aimed to explore the mechanism by which circular RNA RNF111 (circ-RNF111) regulates PNI in SACC by targeting the miR-361-5p/high mobility group box 2 (HMGB2) axis. METHOD: Circ-RNF111 and HMGB2 were highly expressed in SACC specimens, while miR-361-5p was underexpressed. Functional experiments showed that ablating circ-RNF111 or promoting miR-361-5p hindered the biological functions and PNI of SACC-LM cells. RESULTS: HMGB2 overexpression induced the reversal of SACC-LM cell biological functions and PNI caused by circ-RNF111 knockout. Furthermore, reduction of circ-RNF111 suppressed PNI in a SACC xenograft model. Circ-RNF111 regulated HMGB2 expression through targeted modulation of miR-361-5p. CONCLUSION: Taken together, circ-RNF111 stimulates PNI in SACC by miR-361-5p/HMGB2 axis and may serve as a potential therapeutic target for SACC.

Asunto(s)

Carcinoma Adenoide Quístico , MicroARNs , Neoplasias de las Glándulas Salivales , Humanos , Carcinoma Adenoide Quístico/genética , Carcinoma Adenoide Quístico/metabolismo , Carcinoma Adenoide Quístico/patología , ARN Circular/genética , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Neoplasias de las Glándulas Salivales/genética , Neoplasias de las Glándulas Salivales/metabolismo , Neoplasias de las Glándulas Salivales/patología , Factores de Transcripción/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Línea Celular Tumoral , Invasividad Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Movimiento Celular/genética , Proliferación Celular , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

CONTEXT: Central precocious puberty (CPP) can have a familial form in approximately one-quarter of the children. The recognition of this inherited condition increased after the identification of autosomal dominant CPP with paternal transmission caused by mutations in the MKRN3 and DLK1 genes. OBJECTIVE: We aimed to characterize the inheritance and estimate the prevalence of familial CPP in a large multiethnic cohort; to compare clinical and hormonal features, as well as treatment response to GnRH analogs (GnRHa), in children with distinct modes of transmission; and to investigate the genetic basis of familial CPP. METHODS: We retrospectively studied 586 children with a diagnosis of CPP. Patients with familial CPP (n = 276) were selected for clinical and genetic analysis. Data from previous studies were grouped, encompassing sequencing of MKRN3 and DLK1 genes in 204 patients. Large-scale parallel sequencing was performed in 48 individuals from 34 families. RESULTS: The prevalence of familial CPP was estimated at 22%, with a similar frequency of maternal and paternal transmission. Pedigree analyses of families with maternal transmission suggested an autosomal dominant inheritance. Clinical and hormonal features, as well as treatment response to GnRHa, were similar among patients with different forms of transmission of familial CPP. MKRN3 loss-of-function mutations were the most prevalent cause of familial CPP, followed by DLK1 loss-of-function mutations, affecting, respectively, 22% and 4% of the studied families; both affected exclusively families with paternal transmission. Rare variants of uncertain significance were identified in CPP families with maternal transmission. CONCLUSION: We demonstrated a similar prevalence of familial CPP with maternal and paternal transmission. MKRN3 and DLK1 loss-of-function mutations were the major causes of familial CPP with paternal transmission.

Asunto(s)

Pubertad Precoz , Masculino , Niño , Humanos , Pubertad Precoz/tratamiento farmacológico , Pubertad Precoz/epidemiología , Pubertad Precoz/genética , Estudios Retrospectivos , Mutación , Padre , Patrón de Herencia , Ubiquitina-Proteína Ligasas/genética , Pubertad

RESUMEN

Neuronal L-type Ca2+ channels of the CaV1.3 subclass are transmembrane protein complexes that contribute to the pacemaker activity in the adult substantia nigra dopaminergic neurons. The altered function of these channels may play a role in the development and progress of neurodegenerative mechanisms implicated in Parkinson's disease (PD). Although L-type channel expression is precisely regulated, an increased functional expression has been observed in PD. Previously, we showed that Parkin, an E3 enzyme of the ubiquitin-proteasome system (UPS) interacts with neuronal CaV2.2 channels promoting their ubiquitin-mediated degradation. In addition, previous studies show an increase in CaV1.3 channel activity in dopaminergic neurons of the SNc and that Parkin expression is reduced in PD. These findings suggest that the decrease in Parkin may affect the proteasomal degradation of CaV1.3, which helps explain the increase in channel activity. Therefore, the present report aims to gain insight into the degradation mechanisms of the neuronal CaV1.3 channel by the UPS. Immunoprecipitation assays showed the interaction between Parkin and the CaV1.3 channels expressed in HEK-293 cells and neural tissues. Likewise, Parkin overexpression reduced the total and membrane channel levels and decreased the current density. Consistent with this, patch-clamp recordings in the presence of an inhibitor of the UPS, MG132, prevented the effects of Parkin, suggesting enhanced channel proteasomal degradation. In addition, the half-life of the pore-forming CaV1.3α1 protein was significantly reduced by Parkin overexpression. Finally, electrophysiological recordings using a PRKN knockout HEK-293 cell line generated by CRISPR/Cas9 showed increased current density. These results suggest that Parkin promotes the proteasomal degradation of CaV1.3, which may be a relevant aspect for the pathophysiology of PD.NEW & NOTEWORTHY The increased expression of CaV1.3 calcium channels is a crucial feature of Parkinson's disease (PD) pathophysiology. However, the mechanisms that determine this increase are not yet defined. Parkin, an enzyme of the ubiquitin-proteasome system, is known to interact with neuronal channels promoting their ubiquitin-mediated degradation. Interestingly, Parkin mutations also play a role in PD. Here, the degradation mechanisms of CaV1.3 channels and their relationship with the pathophysiology of PD are studied in detail.

Asunto(s)

Canales de Calcio Tipo L , Enfermedad de Parkinson , Ubiquitina-Proteína Ligasas , Humanos , Neuronas Dopaminérgicas/metabolismo , Células HEK293 , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo

RESUMEN

Ewing sarcoma is a fusion oncoprotein-driven primary bone tumor. A subset of patients (~10%) with Ewing sarcoma are known to harbor germline variants in a growing number of genes involved in DNA damage repair. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. We thus questioned the impact of BARD1 loss on Ewing cell sensitivity to DNA damage and the Ewing sarcoma transcriptome. We demonstrate that PSaRC318 cells, a novel patient-derived cell line harboring a pathogenic BARD1 variant, are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that BARD1 loss enhances cell sensitivity to PARP inhibition plus radiation. Additionally, RNA-seq analysis revealed that loss of BARD1 results in the upregulation of GBP1 (guanylate-binding protein 1), a protein whose expression is associated with variable response to therapy depending on the adult carcinoma subtype examined. Here, we demonstrate that GBP1 contributes to the enhanced sensitivity of BARD1 deficient Ewing cells to DNA damage. Together, our findings demonstrate the impact of loss-of function mutations in DNA damage repair genes, such as BARD1, on Ewing sarcoma treatment response.

Asunto(s)

Neoplasias Óseas , Tumores Neuroectodérmicos Periféricos Primitivos , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Daño del ADN/genética , Reparación del ADN/genética , Neoplasias Óseas/genética , Poli(ADP-Ribosa) Polimerasas/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas de Unión al GTP/genética , Proteína BRCA1/genética

RESUMEN

BACKGROUND: RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. RESULTS: Here, we show that in addition to the PRC1-component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrate that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we show that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter and globally alter the Arabidopsis transcriptome in a similar manner. CONCLUSIONS: Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation through the interaction with VIM1, and indicates that evolutionary unrelated lncRNAs with potentially conserved structures may exert similar functions by interacting with homolog partners.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , ARN Largo no Codificante , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN/metabolismo , Metilación de ADN , Histonas/metabolismo , Humanos , Ácidos Indolacéticos/metabolismo , Plantas/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

Parkinson's disease (PD) is a common neurodegenerative disease characterized by movement disorders as well as loss of dopaminergic neurons. Moreover, genes affecting mitochondrial function, such as SNCA, Parkin, PINK1, DJ-1 and LRRK2, were demonstrated to be associated with PD and other neurodegenerative disease. Additionally, mitochondrial dysfunction and cellular energy imbalance are common markers found in PD. In this study, we used the pink1 null mutants of Drosophila melanogaster as a Parkinson's disease model to investigate how the energetic pathways and mitochondrial functions change during aging in a PD model. In our study, the loss of the pink1 gene decreased the survival percent and the decreased climbing index during aging in pink1-/- flies. Furthermore, there was an impairment in mitochondrial function demonstrated by a decrease in OXPHOS CI&CII-Linked and ETS CI&CII-Linked in pink1-/- flies at 3, 15 and 30 days of life. Interestingly, OXPHOS CII-Linked and ETS CII-Linked presented decreases only at 15 days of life in pink1-/- flies. Moreover, there was an increase in peroxide (H2O2) levels in pink1-/- flies at 15 and 30 days of life. Loss of the pink1 gene also decreased the activity of citrate synthase (CS) and increased the activity of lactate dehydrogenase (LDH) in pink1-/- flies head. Our results demonstrate a metabolic shift in ATP production in pink1-/- flies, which changed from oxidative to glycolytic pathways from 15 days of age, and is apparently more pronounced in the central nervous system.

Asunto(s)

Proteínas de Drosophila , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Envejecimiento , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Parkinson/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is a mitochondrial outer membrane-anchored protein-containing transmembrane domain in its N- and C-terminal regions, where both are exposed to the cytosol. Interestingly the C-terminal region has a RING finger domain responsible for its E3 ligase activity, as ubiquitin or in SUMOylation, interacting with proteins related to mitochondrial fusion and fission, cell survival, and tumor suppressor process, such as Akt. Therefore, MUL1 is involved in various cellular processes, such as mitochondrial dynamics, inter-organelle communication, proliferation, mitophagy, immune response, inflammation and cell apoptosis. MUL1 is expressed at a higher basal level in the heart, immune system organs, and blood. Here, we discuss the role of MUL1 in mitochondrial dynamics and its function in various pathological models, both in vitro and in vivo. In this context, we describe the role of MUL1 in: (1) the inflammatory response, by regulating NF-κB activity; (2) cancer, by promoting cell death and regulating exonuclear function of proteins, such as p53; (3) neurological diseases, by maintaining communication with other organelles and interacting with proteins to eliminate damaged organelles and; (4) cardiovascular diseases, by maintaining mitochondrial fusion/fission homeostasis. In this review, we summarize the latest advances in the physiological and pathological functions of MUL1. We also describe the different substrates of MUL1, acting as a positive or negative regulator in various pathologies associated with mitochondrial dysfunction. In conclusion, MUL1 could be a potential key target for the development of therapies that focus on ensuring the functionality of the mitochondrial network and, furthermore, the quality control of intracellular components by synchronously modulating the activity of different cellular mechanisms involved in the aforementioned pathologies. This, in turn, will guide the development of targeted therapies.

Asunto(s)

Sumoilación , Ubiquitina-Proteína Ligasas , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación