RESUMEN
RNF213, a susceptibility gene for moyamoya disease, is associated with stress responses to various stressors. We previously reported that Rnf213 knockout (KO) mitigated endoplasmic reticulum (ER) stress-induced diabetes in the Akita mouse model of diabetes. However, the role of RNF213 in ER stress regulation remains unknown. In the present study, RNF213 knockdown significantly inhibited the upregulation of ER stress markers (CHOP and spliced XBP1) by chemical ER stress-inducers in HeLa cells. Levels of SEL1L, a critical molecule in ER-associated degradation (ERAD), were increased by RNF213 knockdown, and SEL1L knockdown prevented the inhibitory effect of RNF213 suppression on ER stress in HeLa cells, indicating SEL1L involvement in this inhibition of ER stress. SEL1L upregulation was also confirmed in pancreatic islets of Rnf213 KO/Akita mice and in Rnf213 KO mouse embryonic fibroblasts. Additionally, RNF213 suppression increased levels of HRD1, which forms a complex with SEL1L to degrade misfolded protein in cells under ER stress. In conclusion, we demonstrate that RNF213 depletion inhibits ER stress possibly through elevation of the SEL1L-HRD1 complex, thereby promoting ERAD in vitro and in vivo.
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Estrés del Retículo Endoplásmico , Enfermedad de Moyamoya , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Estrés del Retículo Endoplásmico/genética , Degradación Asociada con el Retículo Endoplásmico , Fibroblastos/metabolismo , Células HeLa , Humanos , Ratones , Enfermedad de Moyamoya/genética , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia ArribaRESUMEN
The cerebrovascular disorder moyamoya disease (MMD) was first described in 1957 in Japan, and is typically considered to be an Asian-specific disease. However, it is globally recognized as one of the major causes of childhood stroke. Although several monogenic diseases are known to be complicated by Moyamoya angiopathy, the ring finger protein 213 gene (RNF213) was identified as a susceptibility gene for MMD. RNF213 is unusual, because (1) it induces MMD with no other recognizable phenotypes, (2) the RNF213 p.R4810K variant is an Asian founder mutation common to Japanese, Korean and Chinese with carrier rates of 0.5-2% of the general population but a low penetrance, and (3) it encodes a relatively largest proteins with a dual AAA+ ATPase and E3 Ligase activities. In this review, we focus on the genetics and genetic epidemiology of RNF213, the pathology of RNF213 R4810K, and the molecular functions of RNF213, and also address the public health contributions to current unresolved issues of MMD. We also emphasize the importance of a more updated definition for MMD, of qualified cohort studies based on genetic epidemiology and an awareness of the ethical issues associated with genetic testing of carriers.
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Adenosina Trifosfatasas/genética , Enfermedad de Moyamoya/genética , Enfermedad de Moyamoya/patología , Ubiquitina-Proteína Ligasas/genética , Adenosina Trifosfatasas/metabolismo , Medición de Riesgo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Autosomal-dominant spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders. In this study, we performed genetic analysis of a unique form of SCA (SCA36) that is accompanied by motor neuron involvement. Genome-wide linkage analysis and subsequent fine mapping for three unrelated Japanese families in a cohort of SCA cases, in whom molecular diagnosis had never been performed, mapped the disease locus to the region of a 1.8 Mb stretch (LOD score of 4.60) on 20p13 (D20S906-D20S193) harboring 37 genes with definitive open reading frames. We sequenced 33 of these and observed a large expansion of an intronic GGCCTG hexanucleotide repeat in NOP56 and an unregistered missense variant (Phe265Leu) in C20orf194, but we found no mutations in PDYN and TGM6. The expansion showed complete segregation with the SCA phenotype in family studies, whereas Phe265Leu in C20orf194 did not. Screening of the expansions in the SCA cohort cases revealed four additional occurrences, but none were revealed in the cohort of 27 Alzheimer disease cases, 154 amyotrophic lateral sclerosis cases, or 300 controls. In total, nine unrelated cases were found in 251 cohort SCA patients (3.6%). A founder haplotype was confirmed in these cases. RNA foci formation was detected in lymphoblastoid cells from affected subjects by fluorescence in situ hybridization. Double staining and gel-shift assay showed that (GGCCUG)n binds the RNA-binding protein SRSF2 but that (CUG)(6) does not. In addition, transcription of MIR1292, a neighboring miRNA, was significantly decreased in lymphoblastoid cells of SCA patients. Our finding suggests that SCA36 is caused by hexanucleotide repeat expansions through RNA gain of function.
Asunto(s)
Intrones/genética , Neuronas Motoras/patología , Proteínas Nucleares/genética , Ataxias Espinocerebelosas/genética , Adulto , Edad de Inicio , Anciano , Enfermedad de Alzheimer/genética , Esclerosis Amiotrófica Lateral/genética , Estudios de Cohortes , Encefalinas/genética , Encefalinas/metabolismo , Femenino , Haplotipos , Humanos , Hibridación Fluorescente in Situ , Escala de Lod , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Mutación Missense , Proteínas Nucleares/metabolismo , Linaje , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Ataxias Espinocerebelosas/clasificación , Ataxias Espinocerebelosas/patologíaRESUMEN
Moyamoya disease (MMD) and moyamoya syndrome are vasculopathies characterized by progressive stenosis in the circle of Willis and its branches. The RNF213 gene, which encodes a novel class of proteins, characterized by both E3 ligase and AAA+ATPase activities, has been identified as the susceptibility gene for MMD. However, its physiological functions remain unknown. MMD and moyamoya syndrome are often accompanied by diabetes mellitus. In this study, we generated Rnf213 knockout (KO) C57BL/6 mice (Rnf213(-/-); Ins2(+/+)), which were mated with Akita (C57BL/6 Rnf213(+/+); Ins2(+/C96Y)) mice, a strain that develops diabetes spontaneously by 5 weeks of age, to obtain mice lacking Rnf213 and carrying the Akita mutation (KO/Akita, Rnf213(-/-); Ins2(+/C96Y)). Body weight and blood glucose concentration were measured from 6 to 20 weeks. Glucose tolerance, insulin resistance, plasma insulin and leptin concentrations, food consumption, pancreatic insulin content and histopathology were evaluated at 18 weeks of age. We found that glucose tolerance, as indicated by AUC, was 20% lower (p<0.05) and insulin contents in pancreas were 150% higher (p<0.05), in KO/Akita than in Akita mice. The number of CHOP positive ß-cells assayed by histopathological examination was 30% lower and food consumption was 34% lower in KO/Akita than in Akita mice (p<0.05 each). These findings indicated that the disruption of Rnf213 improved glucose tolerance by protecting islet ß cells.
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Glucemia/metabolismo , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/patología , Células Secretoras de Insulina/metabolismo , Enfermedad de Moyamoya/complicaciones , Ubiquitina-Proteína Ligasas/fisiología , Adenosina Trifosfatasas , Animales , Peso Corporal/genética , Diabetes Mellitus Tipo 1/etiología , Progresión de la Enfermedad , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Células Secretoras de Insulina/ultraestructura , Leptina/sangre , Ratones , Ratones Noqueados , Enfermedad de Moyamoya/genética , Factor de Transcripción CHOP/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the Circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. In the present study, we characterized phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cell cycle to investigate the mechanism of proliferation inhibition. Overexpression of RNF213 R4810K in HeLa cells inhibited cell proliferation and extended the time of mitosis 4-fold. Ablation of spindle checkpoint by depletion of mitotic arrest deficiency 2 (MAD2) did not shorten the time of mitosis. Mitotic morphology in HeLa cells revealed that MAD2 colocalized with RNF213 R4810K. Immunoprecipitation revealed an RNF213/MAD2 complex: R4810K formed a complex with MAD2 more readily than RNF213 wild-type. Desynchronized localization of MAD2 was observed more frequently during mitosis in fibroblasts from patients (n=3, 61.0 ± 8.2%) compared with wild-type subjects (n=6, 13.1 ± 7.7%; p<0.01). Aneuploidy was observed more frequently in fibroblasts (p<0.01) and induced pluripotent stem cells (iPSCs) (p<0.03) from patients than from wild-type subjects. Vascular endothelial cells differentiated from iPSCs (iPSECs) of patients and an unaffected carrier had a longer time from prometaphase to metaphase than those from controls (p<0.05). iPSECs from the patients and unaffected carrier had significantly increased mitotic failure rates compared with controls (p<0.05). Thus, RNF213 R4810K induced mitotic abnormalities and increased risk of genomic instability.
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Predisposición Genética a la Enfermedad , Variación Genética , Inestabilidad Genómica , Mitosis/genética , Enfermedad de Moyamoya/genética , Ubiquitina-Proteína Ligasas/genética , Adenosina Trifosfatasas , Genotipo , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Fenotipo , Células Madre Pluripotentes/metabolismoRESUMEN
Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. Induced pluripotent stem cells (iPSCs) were established from unaffected fibroblast donors with wild-type RNF213 alleles, and from carriers/patients with one or two RNF213 R4810K alleles. Angiogenic activities of iPSC-derived vascular endothelial cells (iPSECs) from patients and carriers were lower (49.0 ± 19.4%) than from wild-type subjects (p<0.01). Gene expression profiles in iPSECs showed that Securin was down-regulated (p<0.01) in carriers and patients. Overexpression of RNF213 R4810K downregulated Securin, inhibited angiogenic activity (36.0 ± 16.9%) and proliferation of humanumbilical vein endothelial cells (HUVECs) while overexpression of RNF213 wild type did not. Securin expression was downregulated using RNA interference techniques, which reduced the level of tube formation in iPSECs and HUVECs without inhibition of proliferation. RNF213 R4810K reduced angiogenic activities of iPSECs from patients with MMD, suggesting that it is a promising in vitro model for MMD.
Asunto(s)
Células Endoteliales/metabolismo , Enfermedad de Moyamoya/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/fisiopatología , Células Madre Pluripotentes/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfatasas , Células Cultivadas , Niño , Regulación hacia Abajo , Células Endoteliales/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad de Moyamoya/patología , Mutación/genética , Neovascularización Patológica/patología , Células Madre Pluripotentes/patología , Securina , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a +TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure.
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Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mutación/fisiología , Proteínas Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Regulación hacia Abajo , Microtúbulos/química , Modelos Moleculares , Datos de Secuencia Molecular , Organismos Modificados Genéticamente , Fosforilación/genética , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Homología de Secuencia de AminoácidoRESUMEN
Here, we present a protocol combining co-immunoprecipitation (Co-IP) and immunofluorescence approaches with cell cycle stage synchronization to detect cell-cycle-specific complexes. We describe steps to synchronize cells at specific cell cycle stages using drugs. We then detail the preparation of cell extracts from synchronized cells and fractionation of the protein complexes with density centrifugation, followed by Co-IP with specific antibodies. Protein-protein interactions are confirmed by localization using immunofluorescence imaging. This protocol is helpful for visualizing the dynamics of protein complex assembly. For complete details on the use and execution of this protocol, please refer to Habu and Kim (2021).1.
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Anticuerpos , Ciclo Celular , División Celular , Técnicas Inmunológicas , InmunoprecipitaciónRESUMEN
Transgene technology is valuable and helpful in many fields, including basic medical and biological research, biotechnology, and therapy. Recent advances in targeting technology accelerate the production of transgenic plants and animals and the application for gene therapy. To develop the technology, we examine the utility as the new safe harbor locus, l-gulono-γ-lactone oxidase (Gulo) locus in human and mice. We performed experiments in vitro and in vivo knockout and knockin mouse and cell lines to validate their applicability using these loci. The Gulo locus might be good candidates for safe harbor loci for transgenic research.
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Edición Génica , Oxidorreductasas , Animales , Humanos , Lactonas , RatonesRESUMEN
Polζ, a DNA polymerase specialized for translesion DNA synthesis (TLS), is comprised of two subunits, the REV3 catalytic subunit and the REV7 accessory subunit. The human REV7 (hREV7) protein is known to interact with hREV3, hREV1 (another TLS protein) and some other proteins such as ADAM9 (a disintegrin and metalloprotease) and ELK-1 (an Ets-like transcription factor). hREV7 is alternatively termed hMAD2L2, because its primary sequence shows 26% identity to that of hMAD2 that plays crucial roles in spindle assembly checkpoint (SAC) via interactions with hMAD1 or hCDC20. Here, we have investigated the molecular basis for the interactions of hREV7/MAD2L2 and hMAD2 with their binding partners. Our results showed that a short sequence of hREV3 is necessary and sufficient for interaction with hREV7. Surprisingly, hMAD2 also binds to the hREV7-binding sequence in hREV3, whereas hMAD2 does not bind to a similar sequence in ADAM9 or ELK-1 and hREV7 does not bind to the hMAD2-binding sequence in hMAD1 or hCDC20. We discuss how hREV7 and hMAD2 recognize their binding partners, and how hREV3 and hREV7 might be involved in SAC.
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Proteínas Mad2/metabolismo , Proteínas ADAM/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Proteínas de la Membrana/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
The centrosome is a cytoplasmic organelle which duplicates once during each cell cycle, and the presence of excess centrosomes promote chromosome instability through chromosome missegregation following cytokinesis. Ionizing radiation (IR) can induce extra centrosomes by permitting the continuation of CDK2/Cyclin-A/E-mediated centrosome duplication when cells are arrested in the cell cycle after irradiation. The work described here shows that, in addition to IR, extra centrosomes were induced in human U2OS and mouse NIH3T3 cells after treatment with agents which include DNA adduct-forming chemicals: benzopyrene (BP), 4-nitroquinoline 1-oxide (4NQO), a DNA cross linker: cis-diamminedichloro-platinum (cisplatin), topoisomerase inhibitors: camptothecin, etoposide, genistein, and ultra-violet light (UV). These agents were divided into two categories with respect to the regulation of p21, which is an inhibitor of CDK2/Cyclin-A/E: specifically, p21 was up-regulated by an IR exposure and treatment with topoisomerase inhibitors. However, UV, BP, 4NQO and cisplatin down-regulated p21 below basal levels. When cells were irradiated with IR in combination with all of these agents, except genistein, enhanced induction of extra centrosomes was observed, regardless of the nature of p21 expression. Genistein significantly suppressed the frequency of IR-induced extra centrosomes in a dose-dependent manner, and 20µg/ml of genistein reduced this frequency to 66%. Consistent with this, genistein substantially up-regulated p21 expression over the induction caused by IR alone, while other agents down-regulated or marginally affected this. This suggests the inhibitory effect of genistein on the induction of extra centrosomes occurs through the inactivation of CDK2/Cyclin-A/E via p21 up-regulation. This hypothesis is supported by the observation that p21 knockdown with siRNA reduced the activity of CDK2/Cyclin-A/E and restored the enhanced effect of a combined treatment with genistein and IR. These results demonstrate the preventive effect of genistein and a crucial role for p21 in IR-induced excess centrosomes.
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Centrosoma/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Genisteína/farmacología , Radiación Ionizante , Animales , Línea Celular Tumoral , Daño del ADN , Relación Dosis-Respuesta a Droga , Técnicas de Inactivación de Genes , Humanos , Ratones , Fitoestrógenos/farmacología , Regulación hacia ArribaRESUMEN
Moyamoya disease (MMD) is a cryptogenic vascular disorder in the intracranial arteries. RING protein 213 (RNF213) is the susceptibility gene for MMD, and encodes a RING domain and a Walker motif. Herein, we identified UBC13 (UBE2N) as an E2 ubiquitin-conjugating enzyme for RNF213 E3 ubiquitin ligase by yeast two-hybrid screening with a fragment containing RNF213 RING domain as bait, and the immunocomplex of RNF213-UBC13 was detected in vivo. Analysis of the ubiquitin chain on RNF213 by monitoring autoubiquitination showed that RNF213 was autoubiquitinated in a K63 chain fashion, but not in a K48 chain fashion. Finally, this RNF213 ubiquitination in a UBC13-dependent manner was required for cell mobility and invasion activity for HUVEC cells in UBC13 knock-down and ubiquitination-dead RNF213 mutant expressing experiments. These findings demonstrated that RNF213 is a K63-linked E3 ubiquitin ligase, and UBC13 is responsible for RNF213 dependent ubiquitination. The RNF213-UBC13 axis may be associated with angiogenic activity and MMD.
RESUMEN
The Mad2 protein plays a key role in the spindle assembly checkpoint (SAC) function. The SAC pathway delays mitotic progression into anaphase until all kinetochores attach to the spindle during mitosis. The formation of the Mad2-p31comet complex correlates with the completion of spindle attachment and the entry into anaphase during mitosis. Herein, we showed that dynein intermediate chain 2c (DNCI2c)-a subunit of dynein motor protein-forms an immunocomplex with p31comet during mitosis. DNCI2c-knockdown resulted in prolonged mitotic arrest in a Mad2-dependent manner. Furthermore, DNCI2c-knockdown-induced mitotic arrest was not rescued by p31comet overexpression. However, the combination of p31comet overexpression with the mitotic drug treatment reversed the mitotic arrest in DNCI2c-knockdown. Together, these results indicate that the DNCI2c-p31comet complex plays an important role in exiting Mad2-dependent SAC.
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Dineínas/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Dineínas/genética , Células HeLa , Humanos , Proteínas Mad2/metabolismo , Huso Acromático/metabolismoRESUMEN
Modification of the N-terminal tail of histones is required for various nuclear processes. Here, we show that fission yeast Clr6-HDAC (histone deacetylase) regulates the checkpoint kinase Cds1 when DNA replication encounters a stressful condition. We found that the global level of acetylation of histone H4 was constant throughout the normal cell cycle, but was reduced significantly when the cell recovered from the HU-induced cell cycle arrest (or slow DNA replication). We identified the Clr6-HDAC as a component responsible for the reduction in the level of the H4 acetylation. Although DNA replication was completed, the HU-induced cell cycle arrest could not be released even after removal of HU in the clr6-1 mutant. Under this experimental condition, Cds1 kinase was maintained active and remained bound tightly to chromatin. We also demonstrated that Cds1 was active even after treatment with caffeine, an inhibitor for ATM/ATR that is an activator of Cds1. These results indicate that inactivation of Cds1 requires functional Clr6-HDAC independently of the conventional DNA replication checkpoint. When DNA replication is impeded, Clr6-HDAC activity may monitor damage on chromatin structure/environment, which is required for inactivation of Cds1.
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Proteínas de Ciclo Celular/metabolismo , Histona Desacetilasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Acetilación , Ciclo Celular/efectos de los fármacos , Quinasa de Punto de Control 2 , Histonas/metabolismo , Hidroxiurea/farmacología , Schizosaccharomyces/efectos de los fármacosRESUMEN
Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions.
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Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Quinasa 2 Dependiente de la Ciclina/metabolismo , Daño del ADN , Mutágenos/toxicidad , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Estrés Fisiológico , Línea Celular , Humanos , Quinasa Tipo Polo 1RESUMEN
RNF213/Mysterin has been identified as a susceptibility gene for moyamoya disease, a cerebrovascular disease characterized by occlusive lesions in the circle of Willis. The p.R4810K (rs112735431) variant is a founder polymorphism that is strongly associated with moyamoya disease in East Asia. Many non-p.R4810K rare variants of RNF213 have been identified in white moyamoya disease patients, although the ethnic mutations have not been investigated in this population. In the present study, we screened for RNF213 variants in 19 Slovakian and Czech moyamoya disease patients. A total of 69 RNF213 coding exons were directly sequenced in 18 probands and one relative who suffered from moyamoya disease in Slovakia and the Czech Republic. We previously reported one proband harboring RNF213 p.D4013N. Results from the present study identified four rare variants other than p.D4013N (p.R4019C, p.E4042K, p.V4146A, and p.W4677L) in four of the patients. P.V4146A was determined to be a novel de novo mutation, and p.R4019C and p.E4042K were identified as double mutations inherited on the same allele. P.W4677L, found in two moyamoya disease patients and an unaffected subject in the same pedigree, was a rare single nucleotide polymorphism. Functional analysis showed that RNF213 p.D4013N, p.R4019C and p.V4146A-transfected human umbilical vein endothelial cells displayed significant lowered migration, and RNF213 p.V4146A significantly reduced tube formation, indicating that these are disease-causing mutations. Results from the present study identified RNF213 rare variants in 22.2% (4/18 probands) of Slovakian and Czech moyamoya disease patients, confirming that RNF213 may also be a major causative gene in a relative large population of white patients.
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Adenosina Trifosfatasas/genética , Enfermedad de Moyamoya/genética , Ubiquitina-Proteína Ligasas/genética , Población Blanca/genética , Adenosina Trifosfatasas/metabolismo , Adulto , Alelos , Movimiento Celular , Niño , República Checa , Exones , Femenino , Genotipo , Haplotipos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Angiografía por Resonancia Magnética , Masculino , Persona de Mediana Edad , Enfermedad de Moyamoya/patología , Linaje , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Eslovaquia , Ubiquitina-Proteína Ligasas/metabolismo , Adulto JovenRESUMEN
Tumours exist in a hypoxic microenvironment and must limit excessive oxygen consumption. Hypoxia-inducible factor (HIF) controls mitochondrial oxygen consumption, but how/if tumours regulate non-mitochondrial oxygen consumption (NMOC) is unknown. Protein-tyrosine phosphatase-1B (PTP1B) is required for Her2/Neu-driven breast cancer (BC) in mice, although the underlying mechanism and human relevance remain unclear. We found that PTP1B-deficient HER2(+) xenografts have increased hypoxia, necrosis and impaired growth. In vitro, PTP1B deficiency sensitizes HER2(+) BC lines to hypoxia by increasing NMOC by α-KG-dependent dioxygenases (α-KGDDs). The moyamoya disease gene product RNF213, an E3 ligase, is negatively regulated by PTP1B in HER2(+) BC cells. RNF213 knockdown reverses the effects of PTP1B deficiency on α-KGDDs, NMOC and hypoxia-induced death of HER2(+) BC cells, and partially restores tumorigenicity. We conclude that PTP1B acts via RNF213 to suppress α-KGDD activity and NMOC. This PTP1B/RNF213/α-KGDD pathway is critical for survival of HER2(+) BC, and possibly other malignancies, in the hypoxic tumour microenvironment.
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Adenosina Trifosfatasas/metabolismo , Consumo de Oxígeno/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Hipoxia de la Célula , Femenino , Genes erbB-2/genética , Humanos , Ratones , Mitocondrias/metabolismoRESUMEN
BACKGROUND: P.R4810K of RNF213 (mysterin: rs112735431), which is an AAA(+) ATPase, is the susceptibility polymorphism for moyamoya disease (MMD) in East Asians. However, the role of RNF213 R4810K in the etiology of MMD is unknown. METHODS AND RESULTS: To clarify the role of RNF213 in known angiogenic pathways, RNF213 expression was analyzed in endothelial cells (ECs) treated with several angiogenic and antiangiogenic factors, including interferons (IFNs). RNF213 was upregulated by IFN-ß through signal transducer and activator of transcription x in the promoter and mediated antiangiogenic activity of IFN-ß. RNF213 wild-type (WT) overexpression could not lower angiogenesis without IFN-ß, but RNF213 R4810K overexpression could. To correlate biochemical function as ATPase and the role of RNF213 oligomer formation with antiangiogenic activity, we investigated the effects of mutations in the AAA(+) module. A mutation of the Walker B motif (WEQ), which stabilizes oligomerization, inhibited angiogenesis, but AAA(+) module deletion, which cannot initiate oligomerization, did not. Intriguingly, R4810K, similar to WEQ, decreased ATPase activity, suggesting its antiangiogenic activity through stabilizing oligomers. To confirm the antiangiogenic effect of RNF213 upregulation in vivo, vascular EC- or smooth muscle cell-specific Rnf213 R4757K (R4810K ortholog) or WT transgenic (Tg) mice were exposed to hypoxia. Cerebral angiogenesis by hypoxia was suppressed in EC-specific Rnf213 R4757K Tg mice, whereas it was not suppressed in other mice. CONCLUSIONS: This study suggests the importance of inflammatory signals as environmental factors and R4810K carriers for susceptibility to cerebral hypoxia. A specific inhibitor of ATP binding to the first AAA(+) could be a promising therapeutic candidate for MMD.
Asunto(s)
Arterias Cerebrales/enzimología , Células Endoteliales/enzimología , Enfermedad de Moyamoya/enzimología , Neovascularización Fisiológica , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfatasas , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Inductores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Sitios de Unión , Unión Competitiva , Movimiento Celular , Proliferación Celular , Arterias Cerebrales/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Predisposición Genética a la Enfermedad , Células HEK293 , Células HeLa , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Hidrólisis , Hipoxia/complicaciones , Mediadores de Inflamación/farmacología , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedad de Moyamoya/genética , Enfermedad de Moyamoya/fisiopatología , Mutación , Miocitos del Músculo Liso/enzimología , Neovascularización Fisiológica/efectos de los fármacos , Fenotipo , Factores de Tiempo , Transfección , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Highly conserved chromatin assembly factor 1 (CAF-1) is required for histone deposition onto newly synthesized DNA to maintain genome stability. This study shows that the fission yeast Pcf1, the large subunit in CAF-1, is crucial for maintaining checkpoint kinase Cds1. Chromatin recruitment of Cds1 is enhanced by Pcf1 overproduction but is attenuated by the Δpcf1 mutation. Mutation of acetylation sites in the histone H4 tail abrogates the chromatin recruitment of Pcf1, which resembles that of Cds1 as reported previously. The present results provide evidence that chromatin recruitment of Pcf1, moderated by Clr6-HDAC activity, is essential for inactivating Cds1.
RESUMEN
Mad2 is a key component of the spindle assembly checkpoint (SAC) that delays the onset of anaphase until all kinetochores are attached to the spindle. It binds to Cdc20 and prevents it from promoting destruction of an anaphase inhibitor, Securin. Previously, we showed that a Mad2-binding protein, p31(comet), formed a complex with Mad2 upon the completion of spindle attachment. Here, we showed that the overexpression of p31(comet) can abolish the Mad2-dependent SAC that is induced by anti-mitotic drugs, including nocodazole, taxol, and monastrol; these drugs, except monastrol, cause aneuploidy in HeLa cells. In the absence of Eg5, which is a target of monastrol, overexpression of p31(comet) caused premature destruction of Securin and premature sister chromatid separation, but it did not cause aneuploidy. These results indicated that Eg5 kinesin function might be required for checkpoint exit and mitotic progression. Moreover, overexpression of p31(comet) led to resistance against apoptosis that was induced by nocodazole and taxol in human cells, and taxol resistance was dependent on the p31(comet)/Mad2 protein expression level ratio of in cancer cell lines. These results indicated that p31(comet) is an indicator of resistance to anti-mitotic drugs in cancer cells.