RESUMEN
RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.
Asunto(s)
Factores de Transcripción , Transcripción Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Microscopía por Crioelectrón , ADN/metabolismoRESUMEN
Mutations in optineurin (OPTN) have been identified in a small proportion of sporadic and familial amyotrophic lateral sclerosis (ALS) cases. Recent evidences suggest that OPTN would be involved in not only the pathophysiological mechanisms of motor neuron death of ALS but also myofiber degeneration of sporadic inclusion body myositis. However, the detailed role of OPTN in muscle remains unclear. Initially, we showed that OPTN expression levels were significantly increased in the denervated muscles of mice, suggesting that OPTN may be involved in muscle homeostasis. To reveal the molecular role of OPTN in muscle atrophy, we used cultured C2C12 myotubes treated with tumor necrosis factor-like inducer of apoptosis (TWEAK) as an in vitro model of muscle atrophy. Our data showed that OPTN had no effect on the process of muscle atrophy in this model. On the other hand, we found that myogenic differentiation was affected by OPTN. Immunoblotting analysis showed that OPTN protein levels gradually decreased during C2C12 differentiation. Furthermore, OPTN knockdown inhibited C2C12 differentiation, accompanied by reduction of mRNA and protein expression levels of myogenin and MyoD. These findings suggested that OPTN may have a novel function in muscle homeostasis and play a role in the pathogenesis of neuromuscular diseases.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Diferenciación Celular/genética , Ratones , Atrofia Muscular/patología , Proteína MioD/genética , Mioblastos/metabolismo , Miogenina/genética , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismoRESUMEN
Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.
Asunto(s)
Citrus , Humulus , ARN Pequeño no Traducido , Viroides , Viroides/metabolismo , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Corteza de la Planta/metabolismo , Enfermedades de las Plantas/genética , Humulus/genética , Citrus/metabolismoRESUMEN
Optineurin (OPTN) is a multifunctional protein involved in autophagy and secretion, as well as nuclear factor κB (NF-κB) and IRF3 signalling, and OPTN mutations are associated with several human diseases. Here, we show that, in response to viral RNA, OPTN translocates to foci in the perinuclear region, where it negatively regulates NF-κB and IRF3 signalling pathways and downstream pro-inflammatory cytokine secretion. These OPTN foci consist of a tight cluster of small membrane vesicles, which are positive for ATG9A. Disease mutations in OPTN linked to primary open-angle glaucoma (POAG) cause aberrant foci formation in the absence of stimuli, which correlates with the ability of OPTN to inhibit signalling. By using proximity labelling proteomics, we identify the linear ubiquitin assembly complex (LUBAC), CYLD and TBK1 as part of the OPTN interactome and show that these proteins are recruited to this OPTN-positive perinuclear compartment. Our work uncovers a crucial role for OPTN in dampening NF-κB and IRF3 signalling through the sequestration of LUBAC and other positive regulators in this viral RNA-induced compartment, leading to altered pro-inflammatory cytokine secretion.
Asunto(s)
Glaucoma de Ángulo Abierto , Factor de Transcripción TFIIIA , Proteínas de Ciclo Celular , Citocinas/genética , Humanos , Proteínas de Transporte de Membrana , FN-kappa B/genética , FN-kappa B/metabolismo , Transporte de Proteínas , Transducción de Señal , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismoRESUMEN
Optineurin (OPTN) is involved in a variety of mechanisms, such as autophagy, vesicle trafficking, and nuclear factor kappa-B (NF-κB) signaling. Mutations in the OPTN gene have been associated with different pathologies, including glaucoma, amyotrophic lateral sclerosis, and Paget's disease of bone. Since the relationship between fish and mammalian OPTN is not well understood, the objective of the present work was to characterize the zebrafish optn gene and protein structure and to investigate its transcriptional regulation. Through a comparative in silico analysis, we observed that zebrafish optn presents genomic features similar to those of its human counterpart, including its neighboring genes and structure. A comparison of OPTN protein from different species revealed a high degree of conservation in its functional domains and three-dimensional structure. Furthermore, our in vitro transient-reporter analysis identified a functional promoter in the upstream region of the zebrafish optn gene, along with a region important for its transcription regulation. Site-directed mutagenesis revealed that the NF-κB motif is responsible for the activation of this region. In conclusion, with this study, we characterize zebrafish optn and our results indicate that zebrafish can be considered as an alternative model to study OPTN's biological role in bone-related diseases.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Transporte de Membrana , FN-kappa B , Factor de Transcripción TFIIIA , Proteínas de Pez Cebra , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Genómica , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
INTRODUCTION: Optineurin (OPTN)-associated mutations have been implicated in the development of type 12 amyotrophic lateral sclerosis (ALS12). We reported a case of ALS with a new OPTN variant (p.D527fs) and reviewed relevant literature to better understand the phenotypes and pathophysiological mechanisms of ALS12. METHODS: We report a case of a 55-year-old female patient with a new heterozygous variant of the OPTN gene. A literature search of ALS cases associated with the OPTN gene mutations was performed in PubMed with the search criteria as [("amyotrophic lateral sclerosis") OR ("motor neuron disease")] AND ("OPTN"). RESULTS: The case of ALS with a new OPTN variant (p.D527fs) in our report manifested with bulbar involvement in onset and a rapidly progressive course. A literature review of 37 ALS patients with OPTN mutations included 20 males and 16 females with another patient whose gender was not described. The mean onset age of 37 ALS12 patients was 48 with the youngest 23 and the oldest 83 years old. Differences in onset age between male and female patients were not significant. Mean time from initiation to death was 61.8 ± 12.0 months. Patients present with either limb onset (73.5% cases) or bulbar onset (23.5% cases). CONCLUSION: Through the literature review, we summarized the clinical characteristics of ALS12. The phenotypes of the reported patients elucidate the genetic profiles and clinical phenotypes of ALS12. Clinicians should pay close attention to the role of receptor-interacting kinase 1 (RIPK1)-dependent necroptosis in the pathophysiologic development of ALS12, since necroptosis inhibitors are expected as potential therapeutic agents for treating ALS12.
Asunto(s)
Esclerosis Amiotrófica Lateral , Factor de Transcripción TFIIIA , Esclerosis Amiotrófica Lateral/genética , Proteínas de Ciclo Celular/genética , Femenino , Heterocigoto , Humanos , Masculino , Mutación/genética , Fenotipo , Factor de Transcripción TFIIIA/genéticaRESUMEN
Mutations in optineurin, a ubiquitin-binding adaptor protein, cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease of motor neurons linked to chronic inflammation and protein aggregation. The majority of ALS patients, including those carrying the optineurin mutations, exhibit cytoplasmic mislocalization, ubiquitination, and aggregation of nuclear TAR DNA-binding protein 43 kDa (TDP-43). To address the crosstalk between optineurin and TDP-43, we generated optineurin knockout (KO) neuronal and microglial cell lines using the CRISPR/Cas9 approach. Interestingly, we observed that loss of optineurin resulted in elevated TDP-43 protein expression in microglial BV2 but not neuronal Neuro 2a and NSC-34 cell lines. No changes were observed at the mRNA level, suggesting that this increase was post-translationally regulated. To confirm this observation in primary cells, we then used microglia and macrophages from an optineurin loss-of-function mouse model that lacks the C-terminal ubiquitin-binding region (Optn470T), mimicking optineurin truncations in ALS patients. As observed in the BV2 cells, we also found elevated basal levels of TDP-43 protein in Optn470T microglia and bone marrow-derived macrophages. To test if inflammation could further enhance TDP-43 accumulation in cells lacking functional optineurin, we stimulated them with lipopolysaccharide (LPS), and we observed a significant increase in TDP-43 expression following LPS treatment of WT cells. However, this was absent in both BV2 Optn KO and primary Optn470T microglia, which exhibited the same elevated TDP-43 levels as in basal conditions. Furthermore, we did not observe nuclear TDP-43 depletion or cytoplasmic aggregate formation in either Optn470T microglia or LPS-treated WT or Optn470T microglia. Taken together, our results show that optineurin deficiency and insufficiency post-translationally upregulate microglial TDP-43 protein levels and that elevated TDP-43 levels in cells lacking functional optineurin could not be further increased by an inflammatory stimulus, suggesting the presence of a plateau.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Inflamación , Lipopolisacáridos/farmacología , Ratones , Microglía/metabolismo , Mutación , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Ubiquitinas/genéticaRESUMEN
Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Lisosomas/metabolismo , Microglía/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factor de Transcripción TFIIIA/genética , alfa-Sinucleína/metabolismo , Autofagia , Proteínas de Ciclo Celular , Humanos , Proteínas de Transporte de Membrana , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factor de Transcripción TFIIIA/metabolismoRESUMEN
To understand disease mechanisms, a large-scale analysis of human-yeast genetic interactions was performed. Of 1305 human disease genes assayed, 20 genes exhibited strong toxicity in yeast. Human-yeast genetic interactions were identified by en masse transformation of the human disease genes into a pool of 4653 homozygous diploid yeast deletion mutants with unique barcode sequences, followed by multiplexed barcode sequencing to identify yeast toxicity modifiers. Subsequent network analyses focusing on amyotrophic lateral sclerosis (ALS)-associated genes, such as optineurin (OPTN) and angiogenin (ANG), showed that the human orthologs of the yeast toxicity modifiers of these ALS genes are enriched for several biological processes, such as cell death, lipid metabolism, and molecular transport. When yeast genetic interaction partners held in common between human OPTN and ANG were validated in mammalian cells and zebrafish, MAP2K5 kinase emerged as a potential drug target for ALS therapy. The toxicity modifiers identified in this study may deepen our understanding of the pathogenic mechanisms of ALS and other devastating diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , MAP Quinasa Quinasa 5/genética , Ribonucleasa Pancreática/genética , Factor de Transcripción TFIIIA/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/terapia , Animales , Proteínas de Ciclo Celular , Humanos , Proteínas de Transporte de Membrana , Terapia Molecular Dirigida , Proteínas Mutantes/genética , Mutación/genética , Mapas de Interacción de Proteínas/genética , Saccharomyces cerevisiae/genética , Eliminación de Secuencia/genética , Pez Cebra/genéticaRESUMEN
Viroids are circular noncoding RNAs (ncRNAs) that infect plants. Despite differences in the genetic makeup and biogenesis, viroids and various long ncRNAs all rely on RNA structure-based interactions with cellular factors for function. Viroids replicating in the nucleus utilize DNA-dependent RNA polymerase II for transcription, a process that involves a unique splicing form of transcription factor IIIA (TFIIIA-7ZF). Here, we provide evidence showing that potato spindle tuber viroid (PSTVd) interacts with a TFIIIA splicing regulator (ribosomal protein L5 [RPL5]) in vitro and in vivo PSTVd infection compromises the regulatory role of RPL5 over splicing of TFIIIA transcripts, while ectopic expression of RPL5 reduces TFIIIA-7ZF expression and attenuates PSTVd accumulation. Furthermore, we illustrate that the RPL5 binding site on the PSTVd genome resides in the central conserved region critical for replication. Together, our data suggest that viroids can regulate their own replication and modulate specific regulatory factors leading to splicing changes in only one or a few genes. This study also has implications for understanding the functional mechanisms of ncRNAs and elucidating the global splicing changes in various host-pathogen interactions.IMPORTANCE Viroids are the smallest replicons among all living entities. As circular noncoding RNAs, viroids can replicate and spread in plants, often resulting in disease symptoms. Potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids, requires a unique splicing form of transcription factor IIIA (TFIIIA-7ZF) for its propagation. Here, we provide evidence showing that PSTVd directly interacts with a splicing regulator, RPL5, to favor the expression of TFIIIA-7ZF, thereby promoting viroid replication. This finding provides new insights to better understand viroid biology and sheds light on the noncoding RNA-based regulation of splicing. Our discovery also establishes RPL5 as a novel negative factor regulating viroid replication in the nucleus and highlights a potential means for viroid control.
Asunto(s)
ARN no Traducido/fisiología , Proteínas Ribosómicas/metabolismo , Solanum tuberosum/virología , Viroides/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Virus de Plantas/fisiología , ARN Polimerasa II/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Factor de Transcripción TFIIIA/genética , Replicación ViralRESUMEN
Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP PSTVd replication in the nucleoplasm generates (-)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (-)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes.
Asunto(s)
Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , ARN Polimerasa II/metabolismo , ARN no Traducido/genética , Factores de Transcripción/metabolismo , Proteínas de Plantas/genética , ARN Polimerasa II/genética , Nicotiana/genética , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Factores de Transcripción/genéticaRESUMEN
Optineurin (OPTN) was initially identified as a regulator of NF-κB and interferon signaling, but attracted most attention because of its association with various human disorders such as glaucoma, Paget disease of bone, and amyotrophic lateral sclerosis. Importantly, OPTN has recently been identified as an autophagy receptor important for the autophagic removal of pathogens, damaged mitochondria, and protein aggregates. This activity is most likely compromised in patients carrying OPTN mutations, and contributes to the observed phenotypes. In this review we summarize recent studies describing the molecular mechanisms by which OPTN controls immunity and autophagy, and discuss these findings in the context of several diseases that have been associated with OPTN (mal)function.
Asunto(s)
Esclerosis Amiotrófica Lateral/inmunología , Autofagia/genética , Glaucoma/inmunología , Osteítis Deformante/inmunología , Factor de Transcripción TFIIIA/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Proteínas de Ciclo Celular , Glaucoma/genética , Humanos , Inmunidad/genética , Interferones/metabolismo , Proteínas de Transporte de Membrana , Mutación/genética , FN-kappa B/metabolismo , Osteítis Deformante/genética , Transducción de Señal , Factor de Transcripción TFIIIA/genéticaRESUMEN
Mitochondria play an essential role in maintaining cellular homeostasis. The removal of damaged or depolarized mitochondria occurs via mitophagy, in which damaged mitochondria are targeted for degradation via ubiquitination induced by PTEN-induced putative kinase 1 (PINK1) and Parkin. Mitophagy receptors, including optineurin (OPTN), nuclear dot 52 kDa protein (NDP52), and Tax1-binding protein 1 (TAX1BP1), are recruited to mitochondria via ubiquitin binding and mediate autophagic engulfment through their association with microtubule-associated protein light chain 3 (LC3). Here, we use live-cell imaging to demonstrate that OPTN, NDP52, and TAX1BP1 are recruited to mitochondria with similar kinetics following either mitochondrial depolarization or localized generation of reactive oxygen species, leading to sequestration by the autophagosome within â¼45 min after insult. Despite this corecruitment, we find that depletion of OPTN, but not NDP52, significantly slows the efficiency of sequestration. OPTN is phosphorylated by the kinase TANK-binding kinase 1 (TBK1) at serine 177; we find that TBK1 is corecruited with OPTN to depolarized mitochondria. Inhibition or depletion of TBK1, or expression of amyotrophic lateral sclerosis (ALS)-associated OPTN or TBK1 mutant blocks efficient autophagosome formation. Together, these results indicate that although there is some functional redundancy among mitophagy receptors, efficient sequestration of damaged mitochondria in response to mitochondrial stress requires both TBK1 and OPTN. Notably, ALS-linked mutations in OPTN and TBK1 can interfere with mitophagy, suggesting that inefficient turnover of damaged mitochondria may represent a key pathophysiological mechanism contributing to neurodegenerative disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Mitocondrias/metabolismo , Mitofagia , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción TFIIIA/metabolismo , Esclerosis Amiotrófica Lateral/genética , Proteínas de Ciclo Celular , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Transporte de Membrana , Mitocondrias/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción TFIIIA/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Upon stimulation of toll-like receptors with various microbial ligands, induction of a variety of inflammatory genes is elicited by activation of a myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathway. Interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) plays an essential role in this pathway by activating nuclear factor κB (NF-κB) and mitogen-activated kinases (MAPKs). Here, we identified optineurin (OPTN) as an IRAK1-binding protein by yeast two-hybrid screening using IRAK1 as bait. A C-terminal fragment of OPTN harboring a ubiquitin-binding domain was co-immunoprecipitated with IRAK1. In reporter analyses, overexpression of OPTN inhibited IL-1ß-, IRAK1-, and LPS-induced NF-κB activation. Consistently, OPTN deficiency resulted in increased NF-κB activation in response to IL-1ß/LPS stimulation. To address the mechanisms underlying the inhibitory effect of OPTN on NF-κB signaling, we focused on tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), which is an adaptor protein of IRAK1 and upon polyubiquitination plays a crucial role during NF-κB activation. Overexpression of OPTN prevented TRAF6 polyubiquitination. Furthermore, OPTN H486R mutant, which is unable to recruit the deubiquitinase CYLD, failed to inhibit IRAK1-induced NF-κB activation. These results suggest that the IRAK1-binding protein OPTN negatively regulates IL-1ß/LPS-induced NF-κB activation by preventing polyubiquitination of TRAF6.
Asunto(s)
Proteínas del Ojo/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal/fisiología , Factor de Transcripción TFIIIA/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas de Ciclo Celular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Enzima Desubiquitinante CYLD , Proteínas del Ojo/genética , Células HEK293 , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/farmacología , Proteínas de Transporte de Membrana , Ratones , Mutación Missense , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción TFIIIA/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/fisiologíaRESUMEN
BACKGROUND: Fusion genes are good candidates of molecular targets for cancer therapy. However, there is insufficient research on the clinical implications and functional characteristics of fusion genes in colorectal cancer (CRC). METHODS: In this study, we analysed RNA sequencing data of CRC patients (147 tumour and 47 matched normal tissues) to identify oncogenic fusion genes and evaluated their role in CRC. RESULTS: We validated 24 fusion genes, including novel fusions, by three algorithms and Sanger sequencing. Fusions from most patients were mutually exclusive CRC oncogenes and included tumour suppressor gene mutations. Eleven fusion genes from 13 patients (8.8%) were determined as oncogenic fusion genes by analysing their gene expression and function. To investigate their oncogenic impact, we performed proliferation and migration assays of CRC cell lines expressing fusion genes of GTF3A-CDK8, NAGLU- IKZF3, RNF121- FOLR2, and STRN-ALK. Overexpression of these fusion genes increased cell proliferation except GTF3A-CDK8. In addition, overexpression of NAGLU-IKZF3 enhanced migration of CRC cells. We demonstrated that NAGLU-IKZF3, RNF121-FOLR2, and STRN-ALK had tumourigenic effects in CRC. CONCLUSION: In summary, we identified and characterised oncogenic fusion genes and their function in CRC, and implicated NAGLU-IKZF3 and RNF121-FOLR2 as novel molecular targets for personalised medicine development.
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Acetilglucosaminidasa/genética , Neoplasias Colorrectales/genética , Receptor 2 de Folato/genética , Factor de Transcripción Ikaros/genética , Proteínas de la Membrana/genética , Quinasa de Linfoma Anaplásico/genética , Proteínas de Unión a Calmodulina/genética , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Quinasa 8 Dependiente de Ciclina/genética , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Proteínas de Fusión Oncogénica/genética , Medicina de Precisión , Factor de Transcripción TFIIIA/genéticaRESUMEN
Optineurin (OPTN) mutations are linked to glaucoma pathology and E50K mutation shows massive cell death in photoreceptor cells and retinal ganglion cells. However, little is known about E50K-mediated mitochondrial dysfunction in photoreceptor cell degeneration. We here show that overexpression of E50K expression triggered BDNF deficiency, leading to Bax activation in RGC-5â¯cells. BDNF deficiency induced mitochondrial dysfunction by decreasing mitochondrial maximal respiration and reducing intracellular ATP level in RGC-5â¯cells. However, BDNF deficiency did not alter mitochondrial dynamics. Also, BDNF deficiency resulted in LC3-mediated mitophagosome formation in RGC-5â¯cells. These results strongly suggest that E50K-mediated BDNF deficiency plays a critical role in compromised mitochondrial function in glaucomatous photoreceptor cell degeneration.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Factor de Transcripción TFIIIA/genética , Proteína X Asociada a bcl-2/genética , Adenosina Trifosfato/biosíntesis , Sustitución de Aminoácidos , Animales , Factor Neurotrófico Derivado del Encéfalo/deficiencia , Línea Celular , Regulación de la Expresión Génica , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/patología , Mutación , Fosforilación Oxidativa , Fagosomas/metabolismo , Fagosomas/patología , Células Fotorreceptoras de Vertebrados/patología , Ratas , Transducción de Señal , Factor de Transcripción TFIIIA/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Paget's disease of bone (PDB) is the second most frequent metabolic bone disease after osteoporosis. Genetic factors play an important role in PDB, but to date PDB causing mutations were identified only in the Sequestosome 1 gene at the PDB3 locus. OPTN has been recently associated with PDB, however little is known about the effect of genetic variants in this gene in PDB pathophysiology. By sequencing OPTN in SQSTM1 non-carriers PDB patients we found 16 SNPs in regulatory, coding and non-coding regions. One of those was found to be associated with PDB in our cohort - rs2234968. Our results show that rs2238968 effect may be explained by a change in OPTN splicing that give rise to a predicted truncated protein. We also performed functional studies on the variants located in OPTN promoter - rs3829923 and the rare variant -9906 - to investigate putative regulators of OPTN. Our results show that OPTN expression seems to be regulated by SP1, RXR, E47, and the E2F family. In conclusion, our work suggests a potential pathophysiological role of SNPs in OPTN, giving a new perspective about the regulatory mechanisms of this gene. Ultimately we discovered a new variant associated with PDB in OPTN, reinforcing the relevance of this gene for the development of this bone disease.
Asunto(s)
Osteítis Deformante/genética , Polimorfismo de Nucleótido Simple , Factor de Transcripción TFIIIA/genética , Estudios de Casos y Controles , Proteínas de Ciclo Celular , Células Cultivadas , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Masculino , Proteínas de Transporte de Membrana , Osteítis Deformante/patología , Regiones Promotoras Genéticas/genéticaRESUMEN
Optineurin (OPTN) is an autophagy receptor protein that has been implicated in glaucoma and amyotrophic lateral sclerosis. OPTN-mediated autophagy is a complex process involving many autophagy-regulating proteins. Autophagy plays a critical role in removing damaged organelles, intracellular pathogens, and protein aggregates to maintain cellular homeostasis. We identified Ypt1 as a novel interaction partner of OPTN by performing a large-scale yeast-human two-hybrid assay. Coimmunoprecipitation assay showed that OPTN interacted with Rab1, the mammalian homolog of yeast Ypt1, in N2a mouse neuroblastoma cell line. We confirmed this interaction by confocal microscopy showing intracellular colocalization of the two proteins. We observed that a zinc finger domain of OPTN is important for Rab1a binding. Rab1a activity is also required for the binding with OPTN. The role of the OPTN-Rab1a complex in neuronal autophagy was determined by measuring the translocation of microtubule-associated protein light chain 3-EGFP to autophagosomes. In N2a cells, OPTN-induced autophagosome formation was inhibited by Rab1a knockdown, indicating the important role of OPTN-Rab1a interaction in neuronal autophagy processes. Similarly, in N2a cells overexpressing Rab1a, serum starvation-induced formation of autophagosome was enhanced, while OPTN knockdown reduced the Rab1a-induced autophagy. These results show that the OPTN-Rab1a complex modulates autophagosome formation in neuroblastoma cells.
Asunto(s)
Autofagosomas/metabolismo , Proteínas del Ojo/metabolismo , Neuroblastoma/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Autofagia , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proteínas del Ojo/genética , Glaucoma/genética , Glaucoma/metabolismo , Humanos , Proteínas de Transporte de Membrana , Ratones , Células 3T3 NIH , Neuroblastoma/patología , Unión Proteica , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab1/genéticaRESUMEN
Glaucoma is a very common disorder of the eye wherein the disturbance of the structural or functional integrity of the optic nerve causes characteristic atrophic changes in the optic nerve, which may lead to specific visual field defects over time. Primary open angle glaucoma (POAG) is most frequent among the three principle glaucoma subtypes. With well-established role of genes like Myocilin (MYOC), Optineurin (OPTN) and WD repeat Domain 36, (WDR36), at least 29 genetic loci have been found till date to be linked to POAG. Moreover, association studies have found 66 loci with 76 genes associated to POAG till date with conflicting results. This particular study is to summarize the current knowledge regarding the change in glaucoma prevalence worldwide and in India from 1993 onwards and compiles all the studied genes that are involved in POAG pathogenesis in Indian population.
Asunto(s)
Glaucoma de Ángulo Abierto/genética , Mutación , Proteínas de Ciclo Celular , Citocromo P-450 CYP1B1/genética , Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Femenino , Glaucoma de Ángulo Abierto/metabolismo , Glicoproteínas/genética , Humanos , India , Masculino , Proteínas de Transporte de Membrana , Factores de Crecimiento Nervioso/genética , Factor de Transcripción TFIIIA/genética , Población Blanca/genéticaRESUMEN
We have ascertained two families affected with familial amyotrophic lateral sclerosis (ALS) in which they both carry a hexanucleotide repeat expansion in the C9orf72 gene, specifically in individuals who also presented with frontotemporal dementia (FTD) or behavioral variant FTD (bvFTD). While some reports attribute this phenotypic heterogeneity to the C9orf72 expansion alone, we screened for additional genetic variation in known ALS-FTD genes that may also contribute to or modify the phenotypes. We performed genetic testing consisting of C9orf72 hexanucleotide expansion, ATXN2 polyglutamine (polyQ) expansion, and targeted next generation sequencing using the ONDRISeq, a gene panel consisting of 80 genes known to be associated with neurodegenerative diseases such as ALS, FTD, Alzheimer's disease, Parkinson's disease, and vascular cognitive impairment. In addition to the C9orf72 expansion, we observed an ATXN2 polyQ intermediate length expansion, and OPTN p.Met468Arg in patients who exhibited ALS and FTD or bvFTD. We conclude that the C9orf72 expansion likely explains much of the ALS-FTD phenotype; however, inheritance of these additional variants likely modifies the disease course and may provide further evidence for biologically relevant oligogenic inheritance in ALS.