RESUMEN
Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.
Asunto(s)
Proteínas Hierro-Azufre , Humanos , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Masculino , Femenino , Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/enzimología , Enfermedades Neuromusculares/metabolismo , Enfermedades Neuromusculares/patología , Niño , Núcleo Celular/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/genética , Citoplasma/metabolismo , Citoplasma/enzimología , MetalochaperonasRESUMEN
PKC is a multifunctional family of Ser-Thr kinases widely implicated in the regulation of fundamental cellular functions, including proliferation, polarity, motility, and differentiation. Notwithstanding their primary cytoplasmic localization and stringent activation by cell surface receptors, PKC isozymes impel prominent nuclear signaling ultimately impacting gene expression. While transcriptional regulation may be wielded by nuclear PKCs, it most often relies on cytoplasmic phosphorylation events that result in nuclear shuttling of PKC downstream effectors, including transcription factors. As expected from the unique coupling of PKC isozymes to signaling effector pathways, glaring disparities in gene activation/repression are observed upon targeting individual PKC family members. Notably, specific PKCs control the expression and activation of transcription factors implicated in cell cycle/mitogenesis, epithelial-to-mesenchymal transition and immune function. Additionally, PKCs isozymes tightly regulate transcription factors involved in stepwise differentiation of pluripotent stem cells toward specific epithelial, mesenchymal, and hematopoietic cell lineages. Aberrant PKC expression and/or activation in pathological conditions, such as in cancer, leads to profound alterations in gene expression, leading to an extensive rewiring of transcriptional networks associated with mitogenesis, invasiveness, stemness, and tumor microenvironment dysregulation. In this review, we outline the current understanding of PKC signaling "in" and "to" the nucleus, with significant focus on established paradigms of PKC-mediated transcriptional control. Dissecting these complexities would allow the identification of relevant molecular targets implicated in a wide spectrum of diseases.
Asunto(s)
Regulación de la Expresión Génica , Proteína Quinasa C , Transducción de Señal , Regulación de la Expresión Génica/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Factores de Transcripción/metabolismo , Humanos , Animales , Núcleo Celular/enzimología , Núcleo Celular/genéticaRESUMEN
Methylations on nucleosomal histones play fundamental roles in regulating eukaryotic transcription. Jumonji C domain-containing histone demethylases (JMJs) dynamically control the level of histone methylations. However, how JMJ activity is generally regulated is unknown. We found that the tricarboxylic acid cycle-associated enzyme α-ketoglutarate (α-KG) dehydrogenase (KGDH) entered the nucleus, where it interacted with various JMJs to regulate α-KG-dependent histone demethylations by JMJs, and thus controlled genome-wide gene expression in plants. We show that nuclear targeting is regulated by environmental signals and that KGDH is enriched at thousands of loci in Arabidopsis thaliana. Chromatin-bound KGDH catalyzes α-KG decarboxylation and thus may limit its local availability to KGDH-coupled JMJs, inhibiting histone demethylation. Thus, our results uncover a regulatory mechanism for histone demethylations by JMJs.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Histonas , Histona Demetilasas con Dominio de Jumonji , Complejo Cetoglutarato Deshidrogenasa , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/enzimología , Cromatina/metabolismo , Desmetilación , Histona Demetilasas/genética , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
One of the major mysteries in science is how it is possible to pack the cellular chromatin with a total length of over 1 m, into a small sphere with a diameter of 5 mm "the nucleus", and even more difficult to envisage how to make it functional. Although we know that compaction is achieved through the histones, however, the DNA needs to be accessible to the transcription machinery and this is allowed thanks to a variety of very complex epigenetic mechanisms. Either DNA (methylation) or post-translational modifications of histone proteins (acetylation, methylation, ubiquitination and sumoylation) play a crucial role in chromatin remodelling and consequently on gene expression. Recently the serotonylation and dopaminylation of the histone 3, catalyzed by the Transglutaminase type 2 (TG2), has been reported. These novel post-translational modifications catalyzed by a predominantly cytoplasmic enzyme opens a new avenue for future investigations on the enzyme function itself and for the possibility that other biological amines, substrate of TG2, can influence the genome regulation under peculiar cellular conditions. In this review we analyzed the nuclear TG2's biology by discussing both its post-translational modification of various transcription factors and the implications of its epigenetic new face. Finally, we will focus on the potential impact of these events in human diseases.
Asunto(s)
Ensamble y Desensamble de Cromatina , Citoplasma , Epigénesis Genética , Histonas , Transglutaminasas , Humanos , Acetilación , Cromatina , ADN/genética , Metilación de ADN , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Transglutaminasas/genética , Transglutaminasas/metabolismo , Citoplasma/enzimología , Citoplasma/genética , Citoplasma/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiologíaRESUMEN
The cleavage of intracellular domains of receptor-like kinases (RLKs) has an important functional role in the transduction of signals from the cell surface to the nucleus in many organisms. However, the peptidases that catalyze protein cleavage during signal transduction remain poorly understood despite their crucial roles in diverse signaling processes. Here, we report in the flowering plant Arabidopsis thaliana that members of the DA1 family of ubiquitin-regulated Zn metallopeptidases cleave the cytoplasmic kinase domain of transmembrane kinase 1 (TMK1), releasing it for nuclear localization where it represses auxin-responsive cell growth during apical hook formation by phosphorylation and stabilization of the transcriptional repressors IAA32 and IAA34. Mutations in DA1 family members exhibited reduced apical hook formation, and DA1 family-mediated cleavage of TMK1 was promoted by auxin treatment. Expression of the DA1 family-generated intracellular kinase domain of TMK1 by an auxin-responsive promoter fully restored apical hook formation in a tmk1 mutant, establishing the function of DA1 family peptidase activities in TMK1-mediated differential cell growth and apical hook formation. DA1 family peptidase activity therefore modulates TMK1 kinase activity between a membrane location where it stimulates acid cell growth and initiates an auxin-dependent kinase cascade controlling cell proliferation in lateral roots and a nuclear localization where it represses auxin-mediated gene expression and growth.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Núcleo Celular , Proteínas con Dominio LIM , Péptido Hidrolasas , Proteínas Serina-Treonina Quinasas , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/enzimología , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Mutación , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitinas/metabolismoRESUMEN
The dichotomous behavior of superoxide dismutase-2 (SOD2) in cancer biology has long been acknowledged and more recently linked to different posttranslational forms of the enzyme. However, a distinctive activity underlying its tumor-promoting function is yet to be described. Here, we report that acetylation, one of such posttranslational modifications (PTMs), increases SOD2 affinity for iron, effectively changing the biochemical function of this enzyme from that of an antioxidant to a demethylase. Acetylated, iron-bound SOD2 localizes to the nucleus, promoting stem cell gene expression via removal of suppressive epigenetic marks such as H3K9me3 and H3K927me3. Particularly, H3K9me3 was specifically removed from regulatory regions upstream of Nanog and Oct-4, two pluripotency factors involved in cancer stem cell reprogramming. Phenotypically, cells expressing nucleus-targeted SOD2 (NLS-SOD2) have increased clonogenicity and metastatic potential. FeSOD2 operating as H3 demethylase requires H2O2 as substrate, which unlike cofactors of canonical demethylases (i.e., oxygen and 2-oxoglutarate), is more abundant in tumor cells than in normal tissue. Therefore, our results indicate that FeSOD2 is a demethylase with unique activities and functions in the promotion of cancer evolution toward metastatic phenotypes.
Asunto(s)
Neoplasias de la Mama , Núcleo Celular , Histona Demetilasas , Hierro , Células Madre Neoplásicas , Superóxido Dismutasa , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Núcleo Celular/enzimología , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Peróxido de Hidrógeno/metabolismo , Hierro/metabolismo , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Procesamiento Proteico-Postraduccional , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.
Asunto(s)
Núcleo Celular , Motas Nucleares , Proteínas Quinasas , Empalme del ARN , Transporte Activo de Núcleo Celular , Núcleo Celular/enzimología , Células HeLa , Humanos , Proteínas Quinasas/metabolismo , ARN/metabolismo , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina/genéticaRESUMEN
Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.
Asunto(s)
Fosfatidilinositol 3-Quinasas , Proteínas Tirosina Quinasas , Núcleo Celular/enzimología , Células HEK293 , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Multimerización de Proteína , Proteínas Tirosina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Superoxide dismutase 1 (SOD1) is one of the causative genes associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder. SOD1 aggregation contributes to ALS pathogenesis. A fraction of the protein is localized in the nucleus (nSOD1), where it seems to be involved in the regulation of genes participating in the oxidative stress response and DNA repair. Peripheral blood mononuclear cells (PBMCs) were collected from sporadic ALS (sALS) patients (n = 18) and healthy controls (n = 12) to perform RNA-sequencing experiments and differential expression analysis. Patients were stratified into groups with "high" and "low" levels of nSOD1. We obtained different gene expression patterns for high- and low-nSOD1 patients. Differentially expressed genes in high nSOD1 form a cluster similar to controls compared to the low-nSOD1 group. The pathways activated in high-nSOD1 patients are related to the upregulation of HSP70 molecular chaperones. We demonstrated that, in this condition, the DNA damage is reduced, even under oxidative stress conditions. Our findings highlight the importance of the nuclear localization of SOD1 as a protective mechanism in sALS patients.
Asunto(s)
Esclerosis Amiotrófica Lateral/sangre , Esclerosis Amiotrófica Lateral/genética , Núcleo Celular/enzimología , Perfilación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/metabolismo , Leucocitos Mononucleares/metabolismo , ARN/genética , Superóxido Dismutasa-1/metabolismo , Estudios de Casos y Controles , Daño del ADN/genética , Regulación de la Expresión Génica , Ontología de Genes , Histonas/metabolismo , Humanos , Metilación , Análisis de Componente Principal , ARN/metabolismoRESUMEN
The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.
Asunto(s)
Núcleo Celular/enzimología , Proliferación Celular , Replicación del ADN , Exosomas/enzimología , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/enzimología , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Núcleo Celular/genética , Roturas del ADN de Doble Cadena , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Exosomas/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Células 3T3 NIH , Neuroblastoma/genética , Neuroblastoma/patología , Regiones Promotoras Genéticas , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Polimerasa II/genética , Terminación de la Transcripción GenéticaRESUMEN
The cytokine storm is a marker of severity of various diseases and increased mortality. The altered metabolic profile and energy generation of immune cells affects their activation, exacerbating the cytokine storm. Currently, the emerging field of immunometabolism has highlighted the importance of specific metabolic pathways in immune regulation. The glycolytic enzyme pyruvate kinase M2 (PKM2) is a key regulator of immunometabolism and bridges metabolic and inflammatory dysfunction. This enzyme changes its conformation thus walks in different fields including metabolism and inflammation and associates with various transcription factors. This review summarizes the vital role of PKM2 in mediating immunometabolic reprogramming and its role in inducing cytokine storm, with a focus on providing references for further understanding of its pathological functions and for proposing new targets for the treatment of related diseases.
Asunto(s)
Proteínas Portadoras/fisiología , Síndrome de Liberación de Citoquinas/enzimología , Proteínas de la Membrana/fisiología , Hormonas Tiroideas/fisiología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Proteínas Portadoras/agonistas , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Núcleo Celular/enzimología , Citocinas/metabolismo , Células Dendríticas/enzimología , Activación Enzimática , Ferroptosis , Glucólisis , Humanos , Inflamasomas , Inflamación , Células Asesinas Naturales/enzimología , Macrófagos/enzimología , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Estructura Molecular , Oxidación-Reducción , Conformación Proteica , Piroptosis , Hormonas Tiroideas/agonistas , Hormonas Tiroideas/química , Proteínas de Unión a Hormona TiroideRESUMEN
Aconitase, a highly conserved protein across all domains of life, functions in converting citrate to isocitrate in the tricarboxylic acid cycle. Cytosolic aconitase is also known to act as an iron regulatory protein in mammals, binding to the RNA hairpin structures known as iron-responsive elements within the untranslated regions of specific RNAs. Aconitase-2 (Aco2) in fission yeast is a fusion protein consisting of an aconitase and a mitochondrial ribosomal protein, bL21, residing not only in mitochondria but also in cytosol and the nucleus. To investigate the role of Aco2 in the nucleus and cytoplasm of fission yeast, we analyzed the transcriptome of aco2ΔN mutant that is deleted of nuclear localization signal (NLS). RNA sequencing revealed that the aco2ΔN mutation caused increase in mRNAs encoding iron uptake transporters, such as Str1, Str3, and Shu1. The half-lives of mRNAs for these genes were found to be significantly longer in the aco2ΔN mutant than the wild-type strain, suggesting the role of Aco2 in mRNA turnover. The three conserved cysteines required for the catalytic activity of aconitase were not necessary for this role. The UV cross-linking RNA immunoprecipitation analysis revealed that Aco2 directly bound to the mRNAs of iron uptake transporters. Aco2-mediated degradation of iron-uptake mRNAs appears to utilize exoribonuclease pathway that involves Rrp6 as evidenced by genetic interactions. These results reveal a novel role of non-mitochondrial aconitase protein in the mRNA turnover in fission yeast to fine-tune iron homeostasis, independent of regulation by transcriptional repressor Fep1.
Asunto(s)
Aconitato Hidratasa/metabolismo , Proteínas de Transporte de Catión/genética , Regulación Fúngica de la Expresión Génica , Hierro/metabolismo , ARN de Hongos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Transporte de Catión/metabolismo , Núcleo Celular/enzimología , Citoplasma/enzimología , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Factores de Transcripción GATA/genética , Factores de Transcripción GATA/metabolismo , Genes Fúngicos , Proteínas Reguladoras del Hierro/genética , Proteínas Reguladoras del Hierro/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Regulón , Ribonucleasas/genética , Ribonucleasas/metabolismo , Schizosaccharomyces/enzimología , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
BACKGROUND: Aurora-A kinase is important for cellular proliferation and is implicated in the tumorigenesis of several malignancies, including of the ovary. Information regarding the expression patterns of Aurora-A in normal Müllerian epithelium as well as benign, borderline and malignant epithelial ovarian neoplasms is limited. METHODS: We investigated Aurora-A expression by immunohistochemistry in 15 benign, 19 borderline and 17 malignant ovarian serous tumors, and 16 benign, 8 borderline, and 2 malignant ovarian mucinous tumors. Twelve fimbriae from seven patients served as normal Müllerian epithelium controls. We also examined Aurora-A protein expression by western blot in normal fimbriae and tumor specimens. RESULTS: All normal fimbriae (n = 12) showed nuclear but not cytoplasmic Aurora-A immunoreactivity by immunohistochemistry. Benign ovarian tumors also showed strong nuclear Aurora-A immunoreactivity. Forty-eight percent (13/27) of borderline tumors demonstrated nuclear Aurora-A immunoreactivity, while the remainder (52%, 14/27) lacked Aurora-A staining. Nuclear Aurora-A immunoreactivity was absent in all malignant serous tumors, however, 47% (8/17) demonstrated perinuclear cytoplasmic staining. These results were statistically significant when tumor class (benign/borderline/malignant) was compared to immunoreactivity localization or intensity (Fisher Exact Test, p < 0.01). Western blot analysis confirmed the greater nuclear Aurora-A expression in control Müllerian epithelium compared to borderline and malignant tumors. CONCLUSION: Aurora-A kinase is differentially expressed across normal Müllerian epithelium, benign and borderline serous and mucinous ovarian epithelial neoplasms and malignant serous ovarian tumors., with nuclear expression of unphosphorylated Aurora-A being present in normal and benign neoplastic epithelium, and lost in malignant serous neoplasms. Further studies of the possible biological and clinical implications of the loss of nuclear Aurora-A expression in ovarian tumors, and its role in ovarian carcinogenesis are warranted.
Asunto(s)
Aurora Quinasa A/biosíntesis , Carcinoma Epitelial de Ovario/enzimología , Cistadenocarcinoma Mucinoso/enzimología , Cistadenocarcinoma Seroso/enzimología , Ovario/enzimología , Carcinoma Epitelial de Ovario/patología , Núcleo Celular/enzimología , Cistadenocarcinoma Mucinoso/patología , Cistadenocarcinoma Seroso/patología , Citoplasma/enzimología , Epitelio/enzimología , Femenino , HumanosRESUMEN
Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Núcleo Celular/enzimología , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias/enzimología , Proteínas Nucleares/metabolismo , Procesamiento Postranscripcional del ARN , ARN Neoplásico/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Núcleo Celular/genética , ADN Helicasas/genética , Metilación de ADN , Proteínas de Unión al ADN/genética , Células HEK293 , Células HeLa , Humanos , Metilación , Neoplasias/genética , Proteínas Nucleares/genética , Estructuras R-Loop , ARN Neoplásico/genética , Empalmosomas/genética , Empalmosomas/metabolismo , Transcripción Genética , UbiquitinaciónRESUMEN
BACKGROUND: To block repairs of DNA damages, especially the DNA double strand break (DSB) repair, can be used to induce cancer cell death. DSB repair depends on a sequential activation of DNA repair factors that may be potentially targeted for clinical cancer therapy. Up to now, many protein components of DSB repair complex remain unclear or poorly characterized. In this study, we discovered that Transglutaminase 2 (TG2) acted as a new component of DSB repair complex. METHODS: A bioinformatic analysis was performed to identify DNA damage relative genes from dataset from The Cancer Genome Atlas. Immunofluorescence and confocal microscopy were used to monitor the protein localization and recruitment kinetics. Furthermore, immunoprecipitation and mass spectrometry analysis were performed to determine protein interaction of both full-length and fragments or mutants in distinct domain. In situ lung cancer model was used to study the effects cancer therapy in vivo. RESULTS: After DSB induction, cytoplasmic TG2 was extensively mobilized and translocated into nucleus after phosphorylated at T162 site by DNA-PKcs. Nuclear TG2 quickly accumulated at DSB sites and directly interacting with Topoisomerase IIα (TOPOIIα) with its TGase domain to promote DSB repair. TG2 deficient cells lost capacity of DSB repair and become susceptible to ionizing radiation. Specific inhibition of TG2-TOPOIIα interaction by glucosamine also significantly inhibited DSB repair, which increased sensitivity in lung cancer cells and engrafted lung cancers. CONCLUSIONS: These findings elucidate new mechanism of TG2 in DSB repair trough directly interacting with TOPOIIα, inhibition of which provided potential target for overcoming cancer resistance.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN-Topoisomerasas de Tipo II/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Células A549 , Animales , Apoptosis/fisiología , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Línea Celular Tumoral , Núcleo Celular/enzimología , Citoplasma/enzimología , Reparación del ADN , ADN-Topoisomerasas de Tipo II/genética , Minería de Datos , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2/genéticaRESUMEN
Tet3 is the main α-ketoglutarate (αKG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How αKG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into αKG in an NAD+-dependent manner, is redirected to the nucleus by the αKG-consumer protein Tet3, suggesting on-site production of αKG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of αKG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity.
Asunto(s)
Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Dioxigenasas/metabolismo , Glutamato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Neuronas/metabolismo , Animales , Encéfalo/metabolismo , Ciclo del Ácido Cítrico , Dioxigenasas/genética , Epigenómica , Expresión Génica , Glutamato Deshidrogenasa/genética , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Metabolómica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Plasticidad NeuronalRESUMEN
Accumulated evidence shows that OGT-mediated O-GlcNAcylation plays an important role in response to DNA damage repair. However, it is unclear if the "eraser" O-GlcNAcase (OGA) participates in this cellular process. Here, we examined the molecular mechanisms and biological functions of OGA in DNA damage repair, and found that OGA was recruited to the sites of DNA damage and mediated deglycosylation following DNA damage. The recruitment of OGA to DNA lesions is mediated by O-GlcNAcylation events. Moreover, we have dissected OGA using deletion mutants and found that C-terminal truncated OGA including the pseudo HAT domain was required for the recruitment of OGA to DNA lesions. Using unbiased protein affinity purification, we found that the pseudo HAT domain was associated with DNA repair factors including NONO and the Ku70/80 complex. Following DNA damage, both NONO and the Ku70/80 complex were O-GlcNAcylated by OGT. The pseudo HAT domain was required to recognize NONO and the Ku70/80 complex for their deglycosylation. Suppression of the deglycosylation prolonged the retention of NONO at DNA lesions and delayed NONO degradation on the chromatin, which impaired non-homologus end joining (NHEJ). Collectively, our study reveals that OGA-mediated deglycosylation plays a key role in DNA damage repair.
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Antígenos de Neoplasias/metabolismo , Núcleo Celular/enzimología , Daño del ADN , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Hialuronoglucosaminidasa/metabolismo , Autoantígeno Ku/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismo , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Proliferación Celular , Proteínas de Unión al ADN/genética , Glicosilación , Células HEK293 , Histona Acetiltransferasas/genética , Humanos , Hialuronoglucosaminidasa/genética , Autoantígeno Ku/genética , Dominios Proteicos , Proteínas de Unión al ARN/genética , Especificidad por SustratoRESUMEN
Kin of irregular chiasm-like 3 (Kirrel3), a member of the immunoglobulin superfamily, is expressed in the central nervous system during development and in adulthood. It has been reported that Kirrel3 is involved in the axonal fasciculation in the olfactory bulb, the neuronal migration in the pontine nucleus, and the synapse formation in the hippocampal neurons in mice. Although KIRREL3 mutations have been implicated in autism spectrum disorder and intellectual disability in humans, the comprehensive expression pattern of Kirrel3 in the adult brain is not fully understood. To better visualize Kirrel3 expression pattern and to gain insight into the role of Kirrel3 in the brain, we investigated the expression of Kirrel3 in the adult brain of Kirrel3-heterozygous (Kirrel3+/-) mice, in which Kirrel3-expressing cells could be identified by the expression of ß-galactosidase (ß-gal) in the nucleus of cells. The strong expression of ß-gal was observed in the hippocampus, cerebral cortex, olfactory bulb, amygdala, thalamus, and cerebellum. In the hippocampus, ß-gal was detected in the dentate gyrus and in the ventral parts of CA1 and CA3, which are known to be involved in the social recognition memory. Within the cerebral cortex, many cells with ß-gal expression were observed in the olfactory area and auditory area. In the striatum, neurons with ß-gal expression were mainly observed in the ventral striatum. Expression of ß-gal was observed in all layers in the cerebellum and olfactory bulb, except for the olfactory nerve layer. Double-immunofluorescence staining of ß-galactosidase with neuronal markers revealed that ß-gal expression was exclusively detected in neurons. These results suggest that Kirrel3 may be involved in the maintenance of neuronal networks, such as the maintenance of synaptic connectivity and plasticity in the motor, sensory, and cognitive circuits of adult brain.
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Encéfalo/metabolismo , Proteínas de la Membrana/genética , Animales , Núcleo Celular/enzimología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The human CST complex composed of CTC1, STN1, and TEN1 is critically involved in telomere maintenance and homeostasis. Specifically, CST terminates telomere extension by inhibiting telomerase access to the telomeric overhang and facilitates lagging strand fill in by recruiting DNA Polymerase alpha primase (Pol α-primase) to the telomeric C-strand. Here we reveal that CST has a dynamic intracellular localization that is cell cycle dependent. We report an increase in nuclear CST several hours after the initiation of DNA replication, followed by exit from the nucleus prior to mitosis. We identify amino acids of CTC1 involved in Pol α-primase binding and nuclear localization. We conclude, the CST complex does not contain a nuclear localization signal (NLS) and suggest that its nuclear localization is reliant on Pol α-primase. Hypomorphic mutations affecting CST nuclear import are associated with telomere syndromes and cancer, emphasizing the important role of this process in health.
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Núcleo Celular/enzimología , ADN Polimerasa I/metabolismo , ADN Primasa/metabolismo , Homeostasis del Telómero , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Núcleo Celular/genética , ADN Polimerasa I/genética , ADN Primasa/genética , Replicación del ADN , Células HEK293 , Humanos , Mitosis , Complejos Multiproteicos , Mutación , Unión Proteica , Telómero/genética , Proteínas de Unión a Telómeros/genéticaRESUMEN
INTRODUCTION: Schizophrenia is considered one of the most disabling and severe human diseases worldwide. The etiology of schizophrenia is thought to be multifactorial and evidence suggests that DNA methylation can play an important role in underlying pivotal neurobiological alterations of this disorder. Some studies have demonstrated the effects of dietary supplementation as an alternative approach to the prevention of schizophrenia, including folic acid. However, no study has ever investigated the role of such supplementation in altering the DNA methylation system in the context of schizophrenia. OBJECTIVES: The present study aims to investigate the effects of maternal folic acid supplementation at different doses on nuclear methyltransferase activity of adult rat offspring subjected to an animal model schizophrenia induced by ketamine. METHODS: Adult female Wistar rats, (60 days old) received folic acid-deficient diet, control diet, or control diet plus folic acid supplementation (at 5, 10, or 50 mg/kg) during pregnancy and lactation. After reaching adulthood (60 days), the male offspring of these dams were subjected to the animal model of schizophrenia induced by 7 days of ketamine intraperitoneal injection (25 mg/kg). After the 7-day protocol, the activity of nuclear methyltransferase was evaluated in the brains of the offspring. RESULTS: Maternal folic acid supplementation at 50 mg/kg increased methyltransferase activity in the frontal cortex, while 10 mg/kg increased methyltransferase activity in the hippocampus. In the striatum of offspring treated with ketamine, maternal deficient diet, control diet, and folic acid supplementation at 5 mg/kg decreased methyltransferase activity compared to the control group. The folic acid supplementation at 10 and 50 mg/kg reversed this ketamine effect. CONCLUSIONS: Maternal FA deficiency could be related to schizophrenia pathophysiology, while FA supplementation could present a protective effect since it demonstrated persistent effects in epigenetic parameters in adult offspring.