RESUMEN
We present two methods for enhancing the efficiency of mitochondrial DNA (mtDNA) editing in mice with DddA-derived cytosine base editors (DdCBEs). First, we fused DdCBEs to a nuclear export signal (DdCBE-NES) to avoid off-target C-to-T conversions in the nuclear genome and improve editing efficiency in mtDNA. Second, mtDNA-targeted TALENs (mitoTALENs) are co-injected into mouse embryos to cleave unedited mtDNA. We generated a mouse model with the m.G12918A mutation in the MT-ND5 gene, associated with mitochondrial genetic disorders in humans. The mutant mice show hunched appearances, damaged mitochondria in kidney and brown adipose tissues, and hippocampal atrophy, resulting in premature death.
Asunto(s)
ADN Mitocondrial , Enfermedades Mitocondriales , Animales , Citosina , ADN Mitocondrial/genética , Endonucleasas/metabolismo , Edición Génica/métodos , Humanos , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Señales de Exportación Nuclear/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genéticaRESUMEN
The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.
Asunto(s)
Proteínas Nucleares , Fototropinas , Animales , Fototropinas/genética , Fototropinas/metabolismo , Proteínas Nucleares/metabolismo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Señales de Exportación Nuclear/genética , Luz , Avena/genética , Avena/metabolismo , Oxígeno/metabolismo , Mamíferos/metabolismoRESUMEN
TAP is a general mRNA export receptor and is highly conserved among eukaryotes. The nematode Caenorhabditis elegans has another TAP-like protein, NXF-2, but little is known about its function. In this study, we show that NXF-2 is specifically expressed in germ cells and forms a novel granular structure that is different from that of P granules and that NXF-2 granules are anchored to the nuclear periphery in the mitotic region of the hermaphrodite gonad. In contrast, NXF-2 granules are released within the whole cytoplasm in the meiotic region, where the feminization gene tra-2 starts to function. Both inhibition of XPO-1 (an ortholog of the export receptor CRM1) and mutation of the nuclear export signal of NXF-2 caused the release of NXF-2 granules from the nuclear periphery, indicating that anchoring of NXF-2 granules depends on XPO-1 function. Moreover, inhibition of NXF-2 resulted in a substantial nuclear accumulation of the reporter mRNA carrying the tra-2 3'UTR. These results suggest that, together with XPO-1, NXF-2 exports and anchors tra-2 mRNA to the nuclear periphery to avoid precocious translation until the germ cells reach the meiotic region, thereby contributing to the regulation of tra-2 mRNA expression.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Regiones no Traducidas 3' , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Células Germinativas/metabolismo , Proteínas de la Membrana/metabolismo , Señales de Exportación Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Fragile X syndrome (FXS; MIM 300624) is an X-linked genetic disorder characterized by physical abnormalities associated with intellectual disability and a wide spectrum of neurological and psychiatric impairments. FXS occurs more frequently in males, 1 in 5000 males and 1 in 8000 females accounting for 1-2% of overall intellectual disability (ID). In more than 99% of patients, FXS results from expansions of a CGG triplet repeat (>200 in male) of the FMR1 gene. In the last years an increasing number, albeit still limited, of FXS subjects carrying FMR1 mutations including deletions, splicing errors, missense, and nonsense variants was reported. Nevertheless, the studies concerning the functional consequences of mutations in the FMR1 gene are rare so far and, therefore, we do not have sufficient knowledge regarding the genotype/phenotype correlation. We report a child carrying a hemizygous missense FMR1 (NM_002024.5:c.1325G > A p.Arg442Gln) variant, maternally inherited, associated with facial abnormalities, developmental delay, and social and communication deficits assessed with formal neuropsychological tests. The study contributes to highlighting the clinical differences between the CGG triplet repeat dependent phenotype and FMR1variant dependent phenotype and it also confirms the pathogenicity of the variant being reported for the second time in the literature.
Asunto(s)
Trastorno del Espectro Autista , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Discapacidad Intelectual , Trastorno del Espectro Autista/complicaciones , Trastorno del Espectro Autista/genética , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Humanos , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/genética , Masculino , Mutación Missense , Señales de Exportación Nuclear/genética , FenotipoRESUMEN
Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283 to 291 (designated NES2) and amino acids 602 to 608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256 to 274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1 and importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1- and importin α/ß-mediated transport by specific inhibitors (LMB, importazole, and ivermectin) clearly blocked PPV replication. The mutant viruses with deletions of the NESs or NLS motif of NS1 by using reverse genetics could not be rescued, suggesting that the NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/ß-mediated nuclear import pathway, and PPV proliferation was inhibited by blocking NS1 nuclear import or export. IMPORTANCE PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (>50 kDa), it cannot pass through the nuclear pore complex by diffusion alone and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and their dependence on the CRM1 pathway for nuclear export was demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/ß nuclear import pathway.
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Interacciones Huésped-Patógeno , Carioferinas/metabolismo , Infecciones por Parvoviridae/veterinaria , Parvovirus Porcino/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Enfermedades de los Porcinos/metabolismo , Enfermedades de los Porcinos/virología , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Carioferinas/genética , Ratones , Señales de Exportación Nuclear/genética , Unión Proteica , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/genética , Porcinos , Proteínas no Estructurales Virales/genética , Replicación Viral , Proteína Exportina 1RESUMEN
Nucleophosmin (NPM1) mutations occurring in acute myeloid leukemia (AML) (about 50 so far identified) cluster almost exclusively in exon 12 and lead to common changes at the NPM1 mutants C-terminus, i.e., loss of tryptophans 288 and 290 (or 290 alone) and creation of a new nuclear export signal (NES), at the bases of exportin-1(XPO1)-mediated aberrant cytoplasmic NPM1. Immunohistochemistry (IHC) detects cytoplasmic NPM1 and is predictive of the molecular alteration. Besides IHC and molecular sequencing, Western blotting (WB) with anti-NPM1 mutant specific antibodies is another approach to identify NPM1-mutated AML. Here, we show that among 382 AML cases with NPM1 exon 12 mutations, one was not recognized by WB, and describe the discovery of a novel combination of two mutations involving exon 12. This appeared as a conventional mutation A with the known TCTG nucleotides insertion/duplication accompanied by a second event (i.e., an 8-nucleotide deletion occurring 15 nucleotides downstream of the TCTG insertion), resulting in a new C-terminal protein sequence. Strikingly, the sequence included a functional NES ensuring cytoplasmic relocation of the new mutant supporting the role of cytoplasmic NPM1 as critical in AML leukemogenesis.
Asunto(s)
Leucemia Mieloide Aguda , Señales de Exportación Nuclear/genética , Nucleofosmina/genética , Transporte Activo de Núcleo Celular/genética , Anciano , Animales , Células Cultivadas , Citoplasma/metabolismo , Humanos , Inmunohistoquímica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Mutación , Células 3T3 NIH , Nucleofosmina/química , Nucleofosmina/metabolismo , Transporte de Proteínas/genéticaRESUMEN
The cyclic GMP-AMP synthase (cGAS) is the principal DNA sensor, which binds DNA and triggers the type I interferon production. We used ISD45 or inactivated Vaccinia Virus (VACV) to stimulate cGAS and monitored cellular localization by immunofluorescence microscopy, Operetta high-content screening, and cytoplasmic/nuclear fractionation. LocNES server was used to predict cGAS nuclear export signal (NES) sequence and characterized the function by mutagenesis. This protocol provides a prototype of cGAS subcellular distribution or the identification of NES in other proteins. For complete details on the use and execution of this protocol, please refer to Sun et al. Sun et al. (2021).
Asunto(s)
Microscopía Fluorescente/métodos , Biología Molecular/métodos , Señales de Exportación Nuclear/fisiología , Nucleotidiltransferasas/metabolismo , Animales , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Mamíferos , Mutagénesis , Señales de Exportación Nuclear/genética , Nucleotidiltransferasas/genética , Virus Vaccinia/genéticaRESUMEN
Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein ß-arrestin2 (ß-arr2) regulates tumor suppressor p53 by counteracting Mdm2. ß-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. ß-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. ß-arr2 can be SUMOylated, but no information is available on how SUMO may regulate ß-arr2 nucleocytoplasmic shuttling. While we found ß-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and ß-arr2, via a SUMO interaction motif (SIM), that is required for ß-arr2 cytonuclear trafficking. This SIM promotes association of ß-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective ß-arr2 nuclear entry. Mutation of the SIM inhibits ß-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a ß-arr2 SIM nuclear entry checkpoint, coupled with active ß-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.
Asunto(s)
Proteínas Activadoras de GTPasa/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína SUMO-1/genética , Proteína p53 Supresora de Tumor/genética , Arrestina beta 2/genética , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Humanos , Mutación/genética , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Señales de Exportación Nuclear/genética , Transducción de Señal/genética , Sumoilación/genéticaRESUMEN
Fragile X syndrome (FXS) is the most common monogenetic cause of intellectual disability and autism spectrum disorders. Mostly, FXS is caused by transcriptional silencing of the FMR1 gene due to a repeat expansion in the 5' UTR, and consequently lack of the protein product FMRP. However, in rare cases FXS is caused by other types of variants in the FMR1 gene. We describe a missense variant in the FMR1 gene, identified through whole-exome sequencing, in a boy with intellectual disability and behavioral problems. The variant is located in the FMRP's nuclear export signal (NES). We performed expression and localization studies of the variant in hair roots and HEK293 cells. Our results show normal expression but significant retention of the FMRP in the cells' nucleus. This finding suggests a possible FMRP reduction at its essential functional sites in the dendrites and the synaptic compartments and possible interference of other cellular processes in the nucleus. Together, this might lead to a FXS phenotype in the boy.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Discapacidad Intelectual/genética , Mutación Missense/genética , Señales de Exportación Nuclear/genética , Regiones no Traducidas 5'/genética , Línea Celular , Femenino , Síndrome del Cromosoma X Frágil/genética , Células HEK293 , Humanos , Masculino , Fenotipo , Secuenciación del Exoma/métodosRESUMEN
Spastin, a microtubule-severing AAA ATPase, regulates microtubule dynamics and plays important roles in cell division and neurogenesis. Mutations in the spastin-coding gene SPAST lead to neurodegenerative disorders and cause spastic paraplegia type 4. Spastin has two main isoforms, M1 and M87, that differ only in the presence or absence of 86 N-terminal amino acids and have alternative splicing variants that lack exon4. The N-terminal region of M1 contains a hydrophobic domain, nuclear localization signal (NLS), and nuclear export signal (NES), which partly explains the differences in the two isoforms' localization. However, the mechanisms involved in regulating isoform localization, and the effects of localization on spastin functions are not fully understood. We found endogenous M1 and M87 shuttled between the nucleus and cytoplasm during the cell cycle. We identified a NES (amino acids 195-204) that spans the microtubule-interacting and endosomal-trafficking domain and exon4 region. Furthermore, the NES sequence contains both the coiled-coil and exon4 region of spastin isoforms. Highly conserved leucine 195 in exon3 and the two residues in exon4 are crucial for predicted coiled-coil formation. Mutations in NES or leptomycin B treatment reduced cytoplasmic localization and microtubule fragmentation in M87 rather than in M1. Phosphomimetic mutation of threonine 306 adjacent to the NLS (amino acids 309-312) inhibited nuclear transport of M87. Our results indicate that the newly identified NES in the spastin isoforms containing exon4 regulates the subcellular localization of spastin in coordination with NLS controlled by the phosphorylation state of spastin, and is involved in microtubule severing.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Microtúbulos/genética , Paraplejía Espástica Hereditaria/genética , Espastina/genética , Adenosina Trifosfatasas/genética , Ciclo Celular/genética , Núcleo Celular/genética , Células Cultivadas , Citoplasma/genética , Exones/genética , Humanos , Mutación/genética , Señales de Exportación Nuclear/genética , Paraplejía Espástica Hereditaria/metabolismo , Paraplejía Espástica Hereditaria/patologíaRESUMEN
Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload.
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Transporte Activo de Núcleo Celular/genética , Señales de Exportación Nuclear/genética , Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Núcleo Celular/metabolismo , Perfil Genético , Genómica , Proteínas Fluorescentes Verdes , Mutación , Biosíntesis de Proteínas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The E571K mutation of CRM1 is highly prevalent in some cancers, but its mechanism of tumorigenesis is unclear. Glu571 of CRM1 is located in its nuclear export signal (NES)-binding groove, suggesting that binding of select NESs may be altered. We generated HEK 293 cells with either monoallelic CRM1WT/E571K or biallelic CRM1E571K/E571K using CRISPR/Cas9. We also combined analysis of binding affinities and structures of 27 diverse NESs for wild-type and E571K CRM1 with structure-based bioinformatics. While most NESs bind the two CRM1 similarly, NESs from Mek1, eIF4E-transporter, and RPS2 showed >10-fold affinity differences. These NESs have multiple charged side chains binding close to CRM1 position 571, but this feature alone was not sufficient to predict different binding to CRM1(E571K). Consistent with eIF4E-transporter NES binding weaker to CRM1(E571K), eIF4E-transporter was mislocalized in tumor cells carrying CRM1(E571K). This serves as proof of concept that understanding how CRM1(E571K) affects NES binding provides a platform for identifying cargoes that are mislocalized in cancer upon CRM1 mutation. Finally, we showed that large affinity changes seen with some NES peptides (of Mek1 and RPS2) do not always translate to the full-length cargoes, suggesting limitations with current NES prediction methods. Therefore, comprehensive studies like ours are imperative to identify CRM1 cargoes with real pathogenic potential.
Asunto(s)
Carioferinas/genética , Señales de Exportación Nuclear/genética , Receptores Citoplasmáticos y Nucleares/genética , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos/genética , Núcleo Celular/metabolismo , Cristalografía por Rayos X/métodos , Células HEK293 , Humanos , Carioferinas/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Modelos Moleculares , Mutación/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Unión Proteica/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Ribosómicas/metabolismo , Proteína de Unión al GTP ran/genética , Proteína de Unión al GTP ran/metabolismo , Proteína Exportina 1RESUMEN
Replication protein A (RPA), a heterotrimeric complex, is the major single-stranded DNA binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a nuclear export signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.
Asunto(s)
Núcleo Celular/metabolismo , Estadios del Ciclo de Vida , Morfogénesis , Proteína de Replicación A/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Simulación por Computador , Citoplasma/metabolismo , Morfogénesis/genética , Señales de Exportación Nuclear/genética , Señales de Exportación Nuclear/fisiología , Proteína de Replicación A/genética , Trypanosoma cruzi/citologíaRESUMEN
USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Núcleo Celular , Señales de Exportación Nuclear , Transporte Activo de Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Interfase , Señales de Exportación Nuclear/genética , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismoRESUMEN
We previously demonstrated that CRM1, a major nuclear export factor, accumulates at Hox cluster regions to recruit nucleoporin-fusion protein Nup98HoxA9, resulting in robust activation of Hox genes (Oka et al., 2016). However, whether this phenomenon is general to other leukemogenic proteins remains unknown. Here, we show that two other leukemogenic proteins, nucleoporin-fusion SET-Nup214 and the NPM1 mutant, NPM1c, which contains a nuclear export signal (NES) at its C-terminus and is one of the most frequent mutations in acute myeloid leukemia, are recruited to the HOX cluster region via chromatin-bound CRM1, leading to HOX gene activation in human leukemia cells. Furthermore, we demonstrate that this mechanism is highly sensitive to a CRM1 inhibitor in leukemia cell line. Together, these findings indicate that CRM1 acts as a key molecule that connects leukemogenic proteins to aberrant HOX gene regulation either via nucleoporin-CRM1 interaction (for SET-Nup214) or NES-CRM1 interaction (for NPM1c).
Asunto(s)
Carioferinas/genética , Leucemia Mieloide Aguda/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas Nucleares/genética , Receptores Citoplasmáticos y Nucleares/genética , Transporte Activo de Núcleo Celular/genética , Línea Celular Tumoral , Núcleo Celular/genética , Cromatina/genética , Citoplasma/genética , Proteínas de Unión al ADN/genética , Regulación Leucémica de la Expresión Génica/genética , Chaperonas de Histonas/genética , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/patología , Mutación/genética , Señales de Exportación Nuclear/genética , Nucleofosmina , Proteína Exportina 1RESUMEN
Activation and repression of Notch target genes is mediated by transcription factor CSL, known as Suppressor of Hairless (Su(H)) in Drosophila and CBF1 or RBPJ in human. CSL associates either with co-activator Notch or with co-repressors such as Drosophila Hairless. The nuclear translocation of transcription factor CSL relies on co-factor association, both in mammals and in Drosophila. The Drosophila CSL orthologue Su(H) requires Hairless for repressor complex formation. Based on its role in transcriptional silencing, H protein would be expected to be strictly nuclear. However, H protein is also cytosolic, which may relate to its role in the stabilization and nuclear translocation of Su(H) protein. Here, we investigate the function of the predicted nuclear localization signals (NLS 1-3) and single nuclear export signal (NES) of co-repressor Hairless using GFP-fusion proteins, reporter assays and in vivo analyses using Hairless wild type and shuttling-defective Hairless mutants. We identify NLS3 and NES to be critical for Hairless function. In fact, HâNLS3 mutant flies match H null mutants, whereas HâNLS3âNES double mutants display weaker phenotypes in agreement with a crucial role for NES in H export. As expected for a transcriptional repressor, Notch target genes are deregulated in HâNLS3 mutant cells, demonstrating nuclear requirement for its activity. Importantly, we reveal that Su(H) protein strictly follows Hairless protein localization. Together, we propose that shuttling between the nucleo-cytoplasmic compartments provides the possibility to fine tune the regulation of Notch target gene expression by balancing of Su(H) protein availability for Notch activation.
Asunto(s)
Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Receptores Notch/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Drosophila/genética , Femenino , Señales de Exportación Nuclear/genética , Señales de Localización Nuclear/genética , Fenotipo , Receptores Notch/genética , Factores de Transcripción/genética , Alas de Animales/crecimiento & desarrolloRESUMEN
Nuclear export signal (NES) motifs function as essential regulators of the subcellular location of proteins by interacting with the major nuclear exporter protein, CRM1. Prediction of NES is of great interest in many aspects of research including cancer, but currently available methods, which are mostly based on the sequence-based approaches, have been suffered from high false positive rates since the NES consensus patterns are quite commonly observed in protein sequences. Therefore, finding a feature that can distinguish real NES motifs from false positives is desired to improve the prediction power, but it is quite challenging when only using the sequence. Here, we provide a comprehensive table for the validated cargo proteins, containing the location of the NES consensus patterns with the disordered propensity plots, known protein domain information, and the predicted secondary structures. It could be useful for determining the most plausible NES region in the context of the whole protein sequence and suggests possibilities for some non-binders of the annotated regions. In addition, using the currently available crystal structures of CRM1 bound to various classes of NES peptides, we adopted, for the first time, the structure-based prediction of the NES motifs bound to the CRM1's binding groove. Combining sequence-based and structure-based predictions, we suggest a novel and more straight-forward approach to identify CRM1-binding NES sequences by analysis of their structural prerequisites and energetic evaluation of the stability at the CRM1's binding site.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/metabolismo , Señales de Exportación Nuclear/fisiología , Transporte Activo de Núcleo Celular/genética , Sitios de Unión , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Señales de Exportación Nuclear/genética , Unión Proteica , Estructura Secundaria de Proteína , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Hypoxia is frequently encountered in the microenvironment of solid tumors. Hypoxia-inducible factors (HIFs), the main effectors of cell response to hypoxia, promote cancer cell survival and progression. HIF-1α, the oxygen-regulated subunit of HIF-1, is often correlated with oncogenesis and represents an attractive therapeutic target. We have previously reported that activation HIF-1α by ERK involves modification of two serine residues and masking of a nuclear export signal (NES), all inside a 43-amino acid domain termed ERK Targeted Domain (ETD). Overexpression of ETD variants including wild-type, phospho-mimetic (SE) or NES-less (IA) mutant forms caused HIF-1 inactivation in two hepatocarcinoma cell lines, while a phospho-deficient (SA) form was ineffective and acted as a sequence-specific negative control. To deliver these ETD forms directly into cancer cells, they were fused to the HIV TAT-sequence and produced as cell-permeable peptides. When the TAT-ETD peptides were added to the culture medium of Huh7 cells, they entered the cells and, with the exception of ETD-SA, accumulated inside the nucleus, caused mislocalization of endogenous HIF-1α to the cytoplasm, significant reduction of HIF-1 activity and inhibition of expression of specific HIF-1, but not HIF-2, gene targets under hypoxia. More importantly, transduced nuclear TAT-ETD peptides restricted migration, impaired colony formation and triggered apoptotic cell death of cancer cells grown under hypoxia, while they produced no effects in normoxic cells. These data demonstrate the importance of ERK-mediated activation of HIF-1 for low oxygen adaptation and the applicability of ETD peptide derivatives as sequence-specific HIF-1 and cancer cell growth inhibitors under hypoxia.
Asunto(s)
Apoptosis/fisiología , Péptidos de Penetración Celular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Hipoxia de la Célula , Línea Celular Tumoral , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/farmacología , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Células Hep G2 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Señales de Exportación Nuclear/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Homología de Secuencia de Aminoácido , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
NOP53 is a tumor suppressor protein located in the nucleolus and is translocated to the cytoplasm during infection by vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1), as shown in our previous study. Cytoplasmic NOP53 interacts with the retinoic acid-inducible gene I (RIG-I) to remove its K63-linked ubiquitination, leading to attenuation of type I interferon IFN-β. In the present study, we found no obvious translocation of NOP53 in infection by a mutant virus lacking ICP4 (HSV-1/d120, replication inadequate). Blocking cytoplasmic translocation of NOP53 by the deletion of its nuclear export sequence (NES) abrogated its ability to support viral replication. These results demonstrated that NOP53 redistribution is related to viral replication. It is interesting that treatment with poly (I:C) or RIG-I-N (a constitutively-active variant) directly induced NOP53 cytoplasmic translocation. To better assess the function of cytoplasmic NOP53 in viral replication, the NOP53-derived protein N3-T, which contains a human immunodeficiency virus (HIV)-derived cell-penetrating Tat peptide at the C-terminal region of N3 (residues 330â»432), was constructed and expressed. The recombinant N3-T protein formed trimers, attenuated the expression of IFN-β and IFN-stimulated genes, as well as decreased the phosphorylation level of interferon regulatory factor 3 (IRF3). Furthermore, N3-T promoted the efficient replication of enveloped and non-enveloped DNA and RNA viruses belonging to 5 families. Our findings expand the understanding of the mechanism by which viruses utilize the nucleolar protein NOP53 for optimal viral replication.
Asunto(s)
Citoplasma/metabolismo , Interacciones Huésped-Patógeno/inmunología , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Replicación Viral , Animales , Línea Celular , Péptidos de Penetración Celular/química , Proteína 58 DEAD Box/genética , Regulación hacia Abajo/efectos de los fármacos , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/genética , Señales de Exportación Nuclear/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Fosforilación/efectos de los fármacos , Poli I-C/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Eliminación de Secuencia , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Glucokinase (GCK) plays a key role in glucose homeostasis. Heterozygous inactivating mutations in the GCK gene cause the familial, mild fasting hyperglycaemia named MODY2. Besides its particular kinetic characteristics, glucokinase is regulated by subcellular compartmentation in hepatocytes. Glucokinase regulatory protein (GKRP) binds to GCK, leading to enzyme inhibition and import into the nucleus at fasting. When glucose concentration increases, GCK-GKRP dissociates and GCK is exported to the cytosol due to a nuclear export signal (NES). With the aim to characterize the GCK-NES, we have functionally analysed nine MODY2 mutations located within the NES sequence. Recombinant GCK mutants showed reduced catalytic activity and, in most cases, protein instability. Most of the mutants interact normally with GKRP, although mutations L306R and L309P impair GCK nuclear import in cotransfected cells. We demonstrated that GCK-NES function depends on exportin 1. We further showed that none of the mutations fully inactivate the NES, with the exception of mutation L304P, which likely destabilizes its α-helicoidal structure. Finally, we found that residue Glu300 negatively modulates the NES activity, whereas other residues have the opposite effect, thus suggesting that some of the NES spacer residues contribute to the low affinity of the NES for exportin 1, which is required for its proper functioning. In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients.