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1.
J Photochem Photobiol B ; 203: 111779, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31927487

RESUMEN

Light is a key environmental factors affecting anthocyanin accumulation in plants. Ubiquitin E3 ligase COP1 has been proved to be a negative regulator involved in light-regulated plant development process, whereas the function and expression specificity of COP1 in anthocyanin biosynthesis in sweet cherry remains unclear. In the present study, we identified a COP1 in sweet cherry, named PacCOP1, it exhibited apparent different expression patterns in red-colored 'Hongdeng' and bi-colored 'Satonishiki', with increasing trend largely in 'Satonishiki', but decreasing trend in 'Hongdeng' after veraison, which was contrary to their variation tendency of anthocyanin content. While the expression abundance of anthocyanin biosynthesis related genes were largely increased after veraison, in accordance with anthocyanin content. Correlation analysis proved that the expression of PacCOP1 was negative correlated with the major genes on anthocyanin accumulation in 'Hongdeng' and 'Satonishiki' fruit, in especial PacDFR, PacANS, PacMYBA and PacbHLH33. Furthermore, over-expression of PacCOP1 in Arabidopsis displayed increased COP1 transcript level with negligible pigmentation and corresponding lower expression level of AtPAP1, AtDFR, AtLDOX, and AtUFGT. These results revealed the negative regular role of PacCOP1 in anthocyanin biosynthesis by repressing the PacMYBA transcription level, followed by down-regulating the structural genes expression abundance, eventually leading to attenuated anthocyanin accumulation in fruits.


Asunto(s)
Antocianinas/biosíntesis , Proteínas de Plantas/metabolismo , Prunus avium/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Filogenia , Pigmentación/genética , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/metabolismo , Prunus avium/metabolismo , Alineación de Secuencia , Ubiquitina-Proteína Ligasas/clasificación , Ubiquitina-Proteína Ligasas/genética
2.
Life Sci ; 244: 117322, 2020 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-31958419

RESUMEN

AIMS: Mitochondrial dysfunction is an early prominent feature of Alzheimer's disease (AD). In the present study, we sought to investigate whether defective mitophagy is tightly related to amyloid-ß (Aß)-induced mitochondrial dysfunction. MAIN METHODS: Immunofluorescence, western blot and transmission electron microscopy were used to examine mitophagy. Mitochondrial membrane potential was assessed using the JC-1 dye. Mitochondrial ROS was detected using MitoSOX™ Red staining. KEY FINDINGS: Aß induced mitochondrial dysfunction in HEK293 cells. Moreover, Aß induced an increase in parkin translocation to mitochondria and led to a drastic reduction in cytosolic parkin. Furthermore, Aß-treated cells displayed a microtubule-associated protein 1 light chain 3 (LC3) punctate pattern and elevated mitochondrial LC3-II levels, suggesting the upregulation of mitophagy. Notably, Aß induced the accumulation of mitochondrial p62, which was associated with impaired mitophagy. In addition, Aß-treated cells exhibited fragmented or swollen mitochondria with severely decreased cristae. We then investigated whether overexpression of parkin could protect cells against Aß-induced mitochondrial dysfunction. Interestingly, parkin overexpression inhibited Aß-induced mitochondrial dysfunction. Besides, parkin overexpression increased cytosolic and mitochondrial parkin levels as well as mitochondrial LC3-II levels in Aß-treated cells. Additionally, parkin overexpression reversed the accumulation of p62 in mitochondria, indicating that parkin overexpression restored impaired mitophagy in Aß-treated cells. Importantly, parkin overexpression remarkably reversed Aß-induced mitochondrial fragmentation. SIGNIFICANCE: Our data demonstrate that overexpression of parkin ameliorates impaired mitophagy and promotes the removal of damaged mitochondria in Aß-treated cells, indicating that upregulation of parkin-mediated mitophagy may be a potential strategy for the therapy of AD.


Asunto(s)
Péptidos beta-Amiloides/efectos adversos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/prevención & control , Ubiquitina-Proteína Ligasas/metabolismo , Células HEK293 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Enfermedades Mitocondriales/etiología , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina-Proteína Ligasas/genética
3.
Gene ; 726: 144186, 2020 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-31647998

RESUMEN

Parkinson's disease (PD) is characterized by progressive death of dopamine producing neurons in the substantia nigra pars compacta of the mid brain. Dysfunction of an E3 ligase protein, Parkin, encoded by PARK2 gene, results in accumulation of misfolded proteins in brain cells which lead to the onset of PD. Parkin is a multi-domain protein consisting of N-terminal ubiquitin-like domain (Ubl) followed by RING0, RING1, In Between Ring (IBR) domain and RING2 domain which is present at the C-terminal end of Parkin protein. Ubl domain is the smallest domain of Parkin and is involved in the binding of Parkin with E2 protein molecule required for proper Ubiquitination and functioning of proteins in the brain. Mutations in the Parkin protein are known to be associated with protein dysfunction leading to PD. This study aims to decipher the characteristics and effects of the different mutations in the Ubl domain by an in-silico analysis. The mutations were collected from PDmutDB and COSMIC databases. The pathogenic impacts of amino-acid mutations on Ubl structure and function were analysed by using various computational tools. Due to lack of proper full-chain structure of the Ubl domain, a homology model of the domain was reconstructed using Discovery Studio 2.5 (DS 2.5) software suite. We found that the mutations A31D, A46P, C59F, A46T, E28K, E49K, R42P, R42S, and Q63K were the most deleterious ones which might be associated with the onset of PD. In order to study the dynamic behaviour of the Parkin Ubl domain in cellular environment, molecular dynamics (MD) simulations were carried out using the wild-type and mutant Ubl domains. Our analyses could predict the cellular dynamics of the mutations and therefore might help in predicting the hitherto unknown molecular mechanism of the disease onset and designing precision medicine for the treatment of PD.


Asunto(s)
Mutación/genética , Enfermedad de Parkinson/genética , Dominios Proteicos/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina/genética , Aminoácidos/genética , Humanos , Polimorfismo de Nucleótido Simple/genética , Estructura Terciaria de Proteína/genética , Ubiquitinación/genética
4.
Cancer Sci ; 111(2): 489-501, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31854042

RESUMEN

The NOTCH2 gene plays a role in the development of many tumors. Deltex E3 ubiquitin ligase 3 (DTX3) was identified as a novel E3 ligase for NOTCH2 and as a potential therapeutic target for esophageal cancer. However, whether DTX3 could regulate NOTCH2 to suppress the progression of esophageal carcinoma remains unknown. In our study, NOTCH2 had higher expression in human esophageal carcinoma cell lines compared to normal human esophageal epithelial cell line, and ablation of NOTCH2 suppressed the proliferation and migration of esophageal carcinoma cells. A novel E3 ligase for NOTCH2 was identified by yeast two-hybrid (Y2H) screening, and DTX3 promoted the ubiquitination and degradation of NOTCH2. Further study showed that DTX3 overexpression suppressed the proliferation and tumorigenicity of human oesophageal carcinoma cells. The analysis of tissue samples from patients revealed that the expression of NOTCH2 was high while the expression of DTX3 was low in esophageal cancer. Furthermore, the expression of DTX3 and NOTCH2 showed a significant negative correlation in human oesophageal cancer samples. Our study suggested that the DTX3-NOTCH2 axis plays an important role in the progression of esophageal cancer, and DTX3 acts as an anti-oncogene in esophageal carcinoma, potentially offering a therapeutic target for esophageal cancer.


Asunto(s)
Neoplasias Esofágicas/patología , Receptor Notch2/química , Receptor Notch2/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Invasividad Neoplásica , Trasplante de Neoplasias , Proteolisis , Transducción de Señal , Ubiquitinación
5.
Medicine (Baltimore) ; 98(44): e17488, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31689751

RESUMEN

BACKGROUND: Dysregulated circular RNAs have been implicated in the pathogenesis of cancer. Recent studies indicate that has_circ_0001649 lowly expressed in multiple types of cancer. The purpose of this study is to investigate the roles of has_circ_0001649 as a diagnostic and prognostic marker for Chinese patients with cancer. METHODS: Adhering to preferred reporting items for systematic reviews and meta-analyses guidelines, systematic literature searches were performed using Pubmed, Embase, and the web of Science to retrieve articles fulfilled all inclusion criteria. The significance of has_circ_0001649 in diagnosis and prognosis of cancer patients were evaluated. Meta-Disc 1. 4 and STATA 12. 0 were used to analyze the data from collected studies. RESULTS: Eleven articles with 761 patients were included in present meta-analysis, of which 4 were about diagnosis, 5 were about prognosis, and 6 were about tumor differentiation grade. For the diagnostic value of has_circ_0001649, the pooled results for sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio with their 95% confidential intervals were 0. 78 (0. 73-0. 83), 0. 75 (0. 70-0. 80), 3. 17 (2. 56-3. 93), 0. 29 (0. 23-0. 36), and 11. 41 (7. 80-16. 7), respectively. The area under the curve of summary receiver operator characteristic was 0.8408 (Q = 0. 7725). Meanwhile, the result showed no obvious publication bias in this analysis for the P-value of Deeks' test was .489. For the prognostic value, the pooled hazard ratio for overall survival was 0.45 (0.324-0.626). Lower expression of has_circ_0001649 was also prone to lower tumor differentiation grade (odds ratio = 2.58, P < .0001). CONCLUSIONS: Has_circ_0001649 could be used as a potential biomarker for diagnosis and prognosis in solid cancer. Further prospective studies are required to validate its clinical application.


Asunto(s)
ADN Helicasas/genética , Neoplasias/diagnóstico , Neoplasias/genética , Ubiquitina-Proteína Ligasas/genética , Grupo de Ascendencia Continental Asiática , Biomarcadores de Tumor , China , Humanos , Clasificación del Tumor , Neoplasias/etnología , Neoplasias/mortalidad , Oportunidad Relativa , Pronóstico , Curva ROC , Sensibilidad y Especificidad
6.
Zhonghua Yan Ke Za Zhi ; 55(11): 806-810, 2019 Nov 11.
Artículo en Chino | MEDLINE | ID: mdl-31715676

RESUMEN

Retinoblastoma, the most frequent malignant intraocular tumor in childhood, is caused by oncogenic mutations in the RB1 tumor suppressor gene. Identification of these mutations in patients is important for genetic counseling and clinical management of relatives at risk, and thus probands are conventionally applied gene detection in developed countries. However, gene diagnosis is still in the elementary period in China. This article reviews the characteristics of retinoblastoma genetics and the current status of genetic testing in China, so as to attract more attention from ophthalmologists and to promote regulated gene diagnosis in clinical work. Not only does good understanding of retinoblastoma genetics support optimal care for retinoblastoma children and their families, but also promotes the development in foundational research. (Chin J Ophthalmol, 2019, 55: 806-810).


Asunto(s)
Pruebas Genéticas , Neoplasias de la Retina/diagnóstico , Retinoblastoma/diagnóstico , Niño , China , Análisis Mutacional de ADN , Genes de Retinoblastoma , Humanos , Mutación , Neoplasias de la Retina/genética , Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/genética , Ubiquitina-Proteína Ligasas/genética
7.
EMBO J ; 38(21): e102361, 2019 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-31613024

RESUMEN

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97-ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97-ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97-ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ataxina-3/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Homeostasis , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatasas/genética , Ataxina-3/genética , Supervivencia Celular , Cromatina/genética , Proteínas de Unión al ADN/genética , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación
8.
Nucleic Acids Res ; 47(16): 8502-8520, 2019 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-31616951

RESUMEN

Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling enzyme with an emerging role in DNA damage response (DDR), but the underlying mechanism remains largely unknown. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1), a key chromatin-associated enzyme responsible for the synthesis of poly(ADP-ribose) (PAR) polymers in mammalian cells, interacts with and PARylates MORC2 at two residues within its conserved CW-type zinc finger domain. Following DNA damage, PARP1 recruits MORC2 to DNA damage sites and catalyzes MORC2 PARylation, which stimulates its ATPase and chromatin remodeling activities. Mutation of PARylation residues in MORC2 results in reduced cell survival after DNA damage. MORC2, in turn, stabilizes PARP1 through enhancing acetyltransferase NAT10-mediated acetylation of PARP1 at lysine 949, which blocks its ubiquitination at the same residue and subsequent degradation by E3 ubiquitin ligase CHFR. Consequently, depletion of MORC2 or expression of an acetylation-defective PARP1 mutant impairs DNA damage-induced PAR production and PAR-dependent recruitment of DNA repair proteins to DNA lesions, leading to enhanced sensitivity to genotoxic stress. Collectively, these findings uncover a previously unrecognized mechanistic link between MORC2 and PARP1 in the regulation of cellular response to DNA damage.


Asunto(s)
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Reparación del ADN , Proteínas de Neoplasias/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Procesamiento Proteico-Postraduccional , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Acetilación/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/química , Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Daño del ADN , Células HEK293 , Humanos , Mutación , Acetiltransferasa E N-Terminal/genética , Acetiltransferasa E N-Terminal/metabolismo , Proteínas de Neoplasias/metabolismo , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteolisis/efectos de los fármacos , Transducción de Señal , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos
9.
Nat Commun ; 10(1): 4779, 2019 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-31636267

RESUMEN

Hyperinflammatory syndromes are life-threatening disorders caused by overzealous immune cell activation and cytokine release, often resulting from defects in negative feedback mechanisms. In the quintessential hyperinflammatory syndrome familial hemophagocytic lymphohistiocytosis (HLH), inborn errors of cytotoxicity result in effector cell accumulation, immune dysregulation and, if untreated, tissue damage and death. Here, we describe a human case with a homozygous nonsense R688* RC3H1 mutation suffering from hyperinflammation, presenting as relapsing HLH. RC3H1 encodes Roquin-1, a posttranscriptional repressor of immune-regulatory proteins such as ICOS, OX40 and TNF. Comparing the R688* variant with the murine M199R variant reveals a phenotypic resemblance, both in immune cell activation, hypercytokinemia and disease development. Mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impeding mRNA decay and dysregulating cytokine production. The results from this unique case suggest that impaired Roquin-1 function provokes hyperinflammation by a failure to quench immune activation.


Asunto(s)
Linfohistiocitosis Hemofagocítica/genética , Proteínas de Unión al ARN/genética , Ubiquitina-Proteína Ligasas/genética , Adolescente , Animales , Codón sin Sentido , Consanguinidad , Ciclosporina/uso terapéutico , Eosinofilia/genética , Eosinofilia/inmunología , Homocigoto , Humanos , Inmunofenotipificación , Inmunosupresores/uso terapéutico , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Linfohistiocitosis Hemofagocítica/tratamiento farmacológico , Linfohistiocitosis Hemofagocítica/inmunología , Masculino , Ratones , Monocitos/inmunología , Receptores OX40/genética , Receptores OX40/inmunología , Receptores OX40/metabolismo , Recurrencia , Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Ubiquitina-Proteína Ligasas/inmunología
10.
Nat Commun ; 10(1): 4790, 2019 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-31636353

RESUMEN

Alterations of Ca2+ homeostasis have been implicated in a wide range of neurodegenerative diseases. Ca2+ efflux from the endoplasmic reticulum into the cytoplasm is controlled by binding of inositol 1,4,5-trisphosphate to its receptor. Activated inositol 1,4,5-trisphosphate receptors are then rapidly degraded by the endoplasmic reticulum-associated degradation pathway. Mutations in genes encoding the neuronal isoform of the inositol 1,4,5-trisphosphate receptor (ITPR1) and genes involved in inositol 1,4,5-trisphosphate receptor degradation (ERLIN1, ERLIN2) are known to cause hereditary spastic paraplegia (HSP) and cerebellar ataxia. We provide evidence that mutations in the ubiquitin E3 ligase gene RNF170, which targets inositol 1,4,5-trisphosphate receptors for degradation, are the likely cause of autosomal recessive HSP in four unrelated families and functionally evaluate the consequences of mutations in patient fibroblasts, mutant SH-SY5Y cells and by gene knockdown in zebrafish. Our findings highlight inositol 1,4,5-trisphosphate signaling as a candidate key pathway for hereditary spastic paraplegias and cerebellar ataxias and thus prioritize this pathway for therapeutic interventions.


Asunto(s)
Degradación Asociada con el Retículo Endoplásmico/genética , Fibroblastos/metabolismo , Neuronas/metabolismo , Paraplejía Espástica Hereditaria/genética , Ubiquitina-Proteína Ligasas/genética , Adolescente , Adulto , Animales , Calcio/metabolismo , Línea Celular Tumoral , Niño , Preescolar , Retículo Endoplásmico/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Transducción de Señal , Piel/citología , Paraplejía Espástica Hereditaria/metabolismo , Pez Cebra
11.
Nat Commun ; 10(1): 4705, 2019 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-31624244

RESUMEN

DNA methylation, repressive histone marks, and PIWI-interacting RNA (piRNA) are essential for the control of retrotransposon silencing in the mammalian germline. However, it remains unknown how these repressive epigenetic pathways crosstalk to ensure retrotransposon silencing in the male germline. Here, we show that UHRF1 is responsible for retrotransposon silencing and cooperates with repressive epigenetic pathways in male germ cells. Conditional loss of UHRF1 in postnatal germ cells causes DNA hypomethylation, upregulation of retrotransposons, the activation of a DNA damage response, and switches in the global chromatin status, leading to complete male sterility. Furthermore, we show that UHRF1 interacts with PRMT5, an arginine methyltransferase, to regulate the repressive histone arginine modifications (H4R3me2s and H3R2me2s), and cooperates with the PIWI pathway during spermatogenesis. Collectively, UHRF1 regulates retrotransposon silencing in male germ cells and provides a molecular link between DNA methylation, histone modification, and the PIWI pathway in the germline.


Asunto(s)
Proteínas Argonauta/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Metilación de ADN , Proteína-Arginina N-Metiltransferasas/genética , Retroelementos/genética , Espermatozoides/metabolismo , Ubiquitina-Proteína Ligasas/genética , Animales , Proteínas Argonauta/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Femenino , Silenciador del Gen , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Unión Proteica , Proteína-Arginina N-Metiltransferasas/metabolismo , Espermatogénesis/genética , Ubiquitina-Proteína Ligasas/metabolismo
12.
Life Sci ; 237: 116919, 2019 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-31610200

RESUMEN

AIMS: Stroke-prone spontaneously hypertensive rats (SHRSP) show significantly lower body weight than normotensive Wistar-Kyoto rats (WKY). Our hypotheses are as follows: weight loss of the skeletal muscle is related to hypertension-related diseases, and muscle hypotrophy is useful as a therapeutic target for hypertension and hypertension-related diseases. In this study, we aimed to investigate the pathophysiological characteristics of muscle hypotrophy in SHRSP to determine the therapeutic target molecule(s). MAIN METHODS: The difference in skeletal muscles in the lower leg between WKY and SHRSP was evaluated mainly through weight/tibial length, histological, gene expression, and protein expression analyses. KEY FINDINGS: SHRSP had a significantly lower weight/tibial length in soleus and gastrocnemius, but not in plantaris and tibialis anterior, indicating that muscles consisting of a relatively high amount of slow muscle fiber were affected. This result was confirmed by the histological analysis of soleus, showing that type I fiber mainly decreased the fiber size. Microarray and protein expression analyses showed that the muscle-specific ubiquitin ligase, muscle RING finger 1 (MuRF1), but not atrogin-1, was highly expressed in soleus, but not in plantaris, in SHRSP. TNF-like weak inducer of apoptosis receptor (TWEAKR) was predicted as a MuRF1 up-regulator by Ingenuity Pathway Analysis and immunostained only in type II fiber in WKY but in both type I and II fibers in SHRSP. SIGNIFICANCE: TWEAKR is a type II-specific receptor in the skeletal muscle. Ectopic TWEAKR expression in type I fiber of SHRSP is most likely involved in slow muscle-specific hypotrophy through MuRF1 overexpression.


Asunto(s)
Hipertensión/patología , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Músculo Liso Vascular/patología , Atrofia Muscular/patología , Accidente Cerebrovascular/patología , Receptor de TWEAK/metabolismo , Animales , Hipertensión/complicaciones , Hipertensión/metabolismo , Masculino , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Liso Vascular/metabolismo , Atrofia Muscular/etiología , Atrofia Muscular/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/metabolismo , Receptor de TWEAK/genética , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
13.
Nat Commun ; 10(1): 4417, 2019 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-31562307

RESUMEN

Alterations in light quality significantly affect plant growth and development. In canopy shade, phytochrome photoreceptors perceive reduced ratios of red to far-red light (R:FR) and initiate stem elongation to enable plants to overtop competitors. This shade avoidance response is achieved via the stabilisation and activation of PHYTOCHROME INTERACTING FACTORs (PIFs) which elevate auxin biosynthesis. UV-B inhibits shade avoidance by reducing the abundance and activity of PIFs, yet the molecular mechanisms controlling PIF abundance in UV-B are unknown. Here we show that the UV-B photoreceptor UVR8 promotes rapid PIF5 degradation via the ubiquitin-proteasome system in a response requiring the N terminus of PIF5. In planta interactions between UVR8 and PIF5 are not observed. We further demonstrate that PIF5 interacts with the E3 ligase COP1, promoting PIF5 stabilisation in light-grown plants. Binding of UVR8 to COP1 in UV-B disrupts this stabilisation, providing a mechanism to rapidly lower PIF5 abundance in sunlight.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Tallos de la Planta/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas Cromosómicas no Histona/genética , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Desarrollo de la Planta/genética , Desarrollo de la Planta/efectos de la radiación , Tallos de la Planta/genética , Tallos de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Estabilidad Proteica , Luz Solar , Ubiquitina-Proteína Ligasas/genética , Rayos Ultravioleta
14.
EMBO J ; 38(20): e101744, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31515872

RESUMEN

In Saccharomyces cerevisiae, the silent information regulator (SIR) proteins Sir2/3/4 form a complex that suppresses transcription in subtelomeric regions and at the homothallic mating-type (HM) loci. Here, we identify a non-canonical BRCA1 C-terminal domain (H-BRCT) in Sir4, which is responsible for tethering telomeres to the nuclear periphery. We show that Sir4 H-BRCT and the closely related Dbf4 H-BRCT serve as selective phospho-epitope recognition domains that bind to a variety of phosphorylated target peptides. We present detailed structural information about the binding mode of established Sir4 interactors (Esc1, Ty5, Ubp10) and identify several novel interactors of Sir4 H-BRCT, including the E3 ubiquitin ligase Tom1. Based on these findings, we propose a phospho-peptide consensus motif for interaction with Sir4 H-BRCT and Dbf4 H-BRCT. Ablation of the Sir4 H-BRCT phospho-peptide interaction disrupts SIR-mediated repression and perinuclear localization. In conclusion, the Sir4 H-BRCT domain serves as a hub for recruitment of phosphorylated target proteins to heterochromatin to properly regulate silencing and nuclear order.


Asunto(s)
Silenciador del Gen , Heterocromatina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Regulación Fúngica de la Expresión Génica , Heterocromatina/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Conformación Proteica , Dominios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/química , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Telómero , Ubiquitina Tiolesterasa/genética , Ubiquitina-Proteína Ligasas/genética
15.
Invest Ophthalmol Vis Sci ; 60(12): 3740-3751, 2019 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-31487745

RESUMEN

Purpose: Chronic oxidative stress is an important mechanism of disease in aging disorders. We do not have a good model to recapitulate AMD and other retinal disorders in which chronic oxidative stress plays an important role. We hypothesized that mice with a combined deficiency in superoxide dismutase 1 (Sod1), DJ-1 (Park-7), and Parkin (Prkn) (triple knock out, TKO) would have an increased level of chronic oxidative stress in the retina, with anatomic and functional consequences just with aging. Methods: Eyes of TKO and B6J control mice were (1) monitored with optical coherence tomography (OCT) and electroretinography (ERG) over time, and (2) collected for oxidative marker protein analysis by ELISA or immunohistochemistry and for transmission electron microscopy studies. Results: TKO mice developed qualitative disruptions in outer retinal layers in OCT by 3 months, increased accumulation of fundus spots and subretinal microglia by 6 months of age, significant retinal thinning by 9 months, and decreased ERG signal by 12 months. Furthermore, we found increased accumulation of the oxidative marker malondialdehyde (MDA) in the retina and increased basal laminal deposits (BLD) and mitochondria number and size in the retinal pigment epithelium of aging TKO mice. Conclusions: TKO mice can serve as a platform to study retinal diseases that involve chronic oxidative stress, including macular degeneration, retinal detachment, and ischemic retinopathies. In order to model each of these diseases, additional disease-specific catalysts or triggers could be superimposed onto the TKO mice. Such studies could provide better insight into disease mechanisms and perhaps lead to new therapeutic approaches.


Asunto(s)
Envejecimiento/fisiología , Proteína Desglicasa DJ-1/deficiencia , Degeneración Retiniana/metabolismo , Superóxido Dismutasa-1/deficiencia , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Biomarcadores/metabolismo , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Mitocondrias/patología , Estrés Oxidativo/fisiología , Proteína Desglicasa DJ-1/genética , Retina/metabolismo , Retina/fisiopatología , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Superóxido Dismutasa-1/genética , Tomografía de Coherencia Óptica , Ubiquitina-Proteína Ligasas/genética
16.
Cell Physiol Biochem ; 53(3): 480-495, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31486323

RESUMEN

BACKGROUND/AIMS: Hypoxia Inducible Factor-1α (HIF-1α) is involved in cancer progression and is stabilized by the chaperone HSP90 (Heat Shock Protein 90), preventing degradation. Previously identified HSP90 inhibitors bind to the N-terminal pocket of HSP90, which blocks binding to HIF-1α and induces HIF-1α degradation. N-terminal inhibitors have failed in the clinic as single therapy treatments partially because they induce a heat shock response. SM molecules are HSP90 inhibitors that bind to the C-terminus of HSP90 and do not induce a heat shock response. The effects of these C-terminal inhibitors on HIF-1α are unreported. METHODS: HCT116, MDA-MB-231, PC3, and HEK293T cells were treated with HSP90 inhibitors. qRT-PCR and western blotting was performed to assess mRNA and protein levels of HIF-1α, HSP- and RACK1-related genes. siRNA was used to knockdown RACK1, while MG262 was used to inhibit proteasome activity. Dimethyloxalylglycine (DMOG) was used to inhibit activity of the prolyl hydroxylases (PHDs). Anti-angiogenic activity of HSP90 inhibitors was assessed using a HUVEC tubule formation assay. RESULTS: We show that SM compounds decrease HIF-1α target expression at the mRNA and protein level under hypoxia in colorectal, breast and prostate cancer cells, leading to cell death, without inducing a heat shock response. Surprisingly, we found that when the C-terminal of HSP90 is inhibited, HIF-1α degradation occurs through the proteasome and prolyl hydroxylases in an oxygen-dependent manner even in very low levels of oxygen (tumor hypoxia levels). RACK1 was not required for proteasomal degradation of HIF-1α. CONCLUSION: Our results suggest that by targeting the C-terminus of HSP90 we can exploit the prolyl hydroxylase and proteasome pathway to induce HIF-1α degradation in hypoxic tumors.


Asunto(s)
Hipoxia de la Célula/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Aminoácidos Dicarboxílicos/metabolismo , Western Blotting , Hipoxia de la Célula/genética , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células HCT116 , Células HEK293 , Proteínas HSP90 de Choque Térmico/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células PC-3 , Prolil Hidroxilasas/genética , Prolil Hidroxilasas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
17.
EMBO J ; 38(20): e101430, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31475738

RESUMEN

E2F7 and E2F8 act as tumor suppressors via transcriptional repression of genes involved in S-phase entry and progression. Previously, we demonstrated that these atypical E2Fs are degraded by APC/CC dh1 during G1 phase of the cell cycle. However, the mechanism driving the downregulation of atypical E2Fs during G2 phase is unknown. Here, we show that E2F7 is targeted for degradation by the E3 ubiquitin ligase SCFcyclin F during G2. Cyclin F binds via its cyclin domain to a conserved C-terminal CY motif on E2F7. An E2F7 mutant unable to interact with SCFcyclin F remains stable during G2. Furthermore, SCFcyclin F can also interact and induce degradation of E2F8. However, this does not require the cyclin domain of SCFcyclin F nor the CY motifs in the C-terminus of E2F8, implying a different regulatory mechanism than for E2F7. Importantly, depletion of cyclin F causes an atypical-E2F-dependent delay of the G2/M transition, accompanied by reduced expression of E2F target genes involved in DNA repair. Live cell imaging of DNA damage revealed that cyclin F-dependent regulation of atypical E2Fs is critical for efficient DNA repair and cell cycle progression.


Asunto(s)
Ciclinas/metabolismo , Reparación del ADN , Factor de Transcripción E2F7/metabolismo , Fase G2/fisiología , Proteolisis , Proteínas Represoras/metabolismo , Puntos de Control del Ciclo Celular , Ciclinas/genética , Daño del ADN , Replicación del ADN , Factor de Transcripción E2F7/genética , Células HeLa , Humanos , Unión Proteica , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
18.
J Exp Clin Cancer Res ; 38(1): 384, 2019 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-31477177

RESUMEN

BACKGROUND: Aberrant activation of Notch signaling has been causally linked to the metastasis of hepatocellular carcinoma (HCC), however the underlying molecular mechanisms are still poorly understood. RING finger protein 187 (RNF187) was recently revealed to be a driver of several cancers, but its expression pattern and biological function in HCC are unknown. METHODS: The expression levels of Notch1 and RNF187 were assessed in two independent cohorts of HCC tissues, and modulation of Notch1 in HCC cells was performed to explore the regulatory role of Notch1 in HCC metastasis. RNA-sequencing (RNA-seq), bioinformatics analysis, luciferase reporter analysis, and chromatin immunoprecipitation assay (ChIP) were used to clarify the relationship between Notch1 signaling and its potential target Ring finger protein 187 (RNF187). Gain- and loss-of-function studies were used to dissect the role of Notch1-RNF187 signaling in promoting HCC metastasis. The impact of Notch1-RNF187 activity in determining clinical prognosis for HCC patients was evaluated by multivariate Cox regression. RESULTS: By RNA-seq, luciferase reporter analysis, and ChIP assay, RNF187 was confirmed to be a direct transcriptional target of Notch1, as Notch1 could activate RNF187 promoter whereas the pro-migratory and pro-invasive effects of Notch1 were significantly attenuated by RNF187 knockdown. Meanwhile, RNF187 silencing could attenuate the Notch1-dependent epithelial-mesenchymal transition (EMT). Moreover, overexpression of RNF187 counteracted the inhibitory effect of Notch1 knockdown on cancer progression. Importantly, HCC patients with high level of hepatic Notch1 expression had shorter disease-free survival (DFS) than those with low level of hepatic Notch1 expression. Furthermore, patients with high level of Notch1 and RNF187 co-expression showed the shortest DFS. The expression level of Notch1 and RNF187 was an independent prognostic factor for HCC. CONCLUSIONS: For the first time we identified that RNF187 is an essential factor for Notch1 to promote invasion and metastasis of HCC. Of highly clinical relevance, we found that activation of Notch1-RNF187 correlates with a worse prognosis of HCC patients. These findings provide a solid foundation for developing novel strategies to tackle HCC metastasis.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Receptor Notch1/metabolismo , Transactivadores/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adulto , Anciano , Biomarcadores de Tumor , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidad , Transición Epitelial-Mesenquimal/genética , Femenino , Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Unión Proteica , Transducción de Señal , Transactivadores/genética , Transcripción Genética , Ubiquitina-Proteína Ligasas/genética
19.
Nat Commun ; 10(1): 3616, 2019 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-31399586

RESUMEN

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFß1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFß1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.


Asunto(s)
Fibrosis/metabolismo , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Proteína Smad2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adolescente , Adulto , Anciano , Animales , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibrosis/genética , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , Cardiopatías/genética , Cardiopatías/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Isoformas de Proteínas , Proteína Smad2/genética , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina-Proteína Ligasas/genética , Adulto Joven
20.
Nat Commun ; 10(1): 3645, 2019 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-31409786

RESUMEN

Chronic mitochondrial stress associates with major neurodegenerative diseases. Recovering stressed mitochondria constitutes a critical step of mitochondrial quality control and thus energy maintenance in early stages of neurodegeneration. Here, we reveal Mul1-Mfn2 pathway that maintains neuronal mitochondrial integrity under stress conditions. Mul1 deficiency increases Mfn2 activity that triggers the first phasic mitochondrial hyperfusion and also acts as an ER-Mito tethering antagonist. Reduced ER-Mito coupling leads to increased cytoplasmic Ca2+ load that activates calcineurin and induces the second phasic Drp1-dependent mitochondrial fragmentation and mitophagy. Overexpressing Mfn2, but not Mfn1, mimics Mul1-deficient phenotypes, while expressing PTPIP51, an ER-Mito anchoring protein, suppresses Parkin-mediated mitophagy. Thus, by regulating mitochondrial morphology and ER-Mito contacts, Mul1-Mfn2 pathway plays an early checkpoint role in maintaining mitochondrial integrity. Our study provides new mechanistic insights into neuronal mitochondrial maintenance under stress conditions, which is relevant to several major neurodegenerative diseases associated with mitochondrial dysfunction and altered ER-Mito interplay.


Asunto(s)
Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Calcio/metabolismo , Retículo Endoplásmico/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/genética , Neuronas/citología , Ubiquitina-Proteína Ligasas/genética
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