9 research outputs found

    Búsqueda de señales de transporte nucleocitoplásmico en la proteína KMT2D

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    [ES] KMT2D es una metiltransferasa que se encarga de transferir un grupo metilo a la histona H3, lo que está relacionado con la activación génica. Esta proteína es esencial en el desarrollo embrionario temprano y, además, se expresa también en numerosos tejidos adultos. Las mutaciones en el gen que codifica para la síntesis de KMT2D en la línea germinal están relacionadas con el síndrome de Kabuki, una enfermedad congénita rara. También se ha visto que las mutaciones somáticas están relacionadas con la formación de tumores, siendo KMT2D una de las proteínas más frecuentemente mutadas en el cáncer. Por su actividad metiltransferasa de histonas, se sabe que KMT2D desarrolla su función en el núcleo. Partiendo de la hipótesis inicial de que KMT2D puede tener señales de transporte nucleocitoplásmico funcionales, se realizaron análisis bioinformáticos para predecir tanto posibles señales de localización nuclear (NLS) como señales de exportación nuclear (NES) en su secuencia. Los ensayos experimentales revelaron que KMT2D tiene una NLS funcional y una NES débil. De este modo, si se identifican mutaciones relacionadas con patologías en las regiones en las que se encuentran estas señales, se podría investigar la relación del transporte alterado de KMT2D con las diferentes patologías

    Identification of nucleocytoplasmic transport signals in Rps15 and Ltv1, two proteins related to small ribosomal subunit export

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    [EN] Ribosomes are indispensable organelles for cell survival. Eukaryotic ribosomes are made up of two subunits (60S and 40S), which are independently exported from the nucleolus to the cytoplasm, where they are finally assembled to form the functional ribosome. Due to their large size, ribosomal subunits need nuclear transport receptors, or karyopherins (such as the export receptor XPO1), to move through the nuclear pore complex. Karyopherins recognize cargo proteins bearing nucleocytoplasmic transport signals (nuclear localization signals (NLSs) or nuclear export signals (NESs)). In addition to karyopherins, nucleocytoplasmic transport of the small ribosomal subunit (40S), requires other factors, such as the ribosomal protein Rps15 and the non-ribosomal protein Ltv1. Due to the role of these proteins in the export of small ribosomal subunit, we hypothesized that they could carry still uncharacterized nucleocytoplasmic transport signals. Here we describe the identification of three new functional nuclear transport signals: one NLS and one NES in Rps15, as well as one NLS in Ltv1. Our results provide new information on the specific amino acid sequences that contribute to the function of Ltv1 and Rps15 as adapters in the nuclear export of the small ribosomal subunit.[ES] Los ribosomas son orgánulos indispensables para la supervivencia de la célula. Los ribosomas eucariotas están compuestos por dos subunidades (60S y 40S), que se exportan desde el nucleolo hasta el citoplasma de forma independiente, donde son finalmente ensambladas para dar lugar al ribosoma funcional. Debido a su gran tamaño, las subunidades ribosómicas necesitan receptores de transporte nuclear, o carioferinas (como el receptor de exportación XPO1), para atravesar el complejo del poro nuclear. Las carioferinas reconocen proteínas “cargo” que portan señales de transporte nucleocitoplásmico (señales de localización nuclear (NLSs) o señales de exportación nuclear (NESs)). Además de las carioferinas, el transporte nucleocitoplásmico de la subunidad ribosómica pequeña (40S), requiere otros factores, como la proteína ribosómica Rps15 y la proteína no ribosómica Ltv1. Debido al importante papel que juegan en la exportación de la subunidad ribosómica pequeña, planteamos la hipótesis de que estas proteínas podrían poseer señales de transporte nucleocitoplásmico aún sin caracterizar. Aquí describimos la identificación de tres nuevas señales de transporte nuclear funcionales: una NLS y una NES en Rps15, así como una NLS en Ltv1. Nuestros resultados aportan nueva información sobre las secuencias de aminoácidos concretas que contribuyen al funcionamiento de Ltv1 y de Rps15 como adaptadores en la exportación nuclear de la subunidad ribosómica pequeña

    Using a Simple Cellular Assay to Map NES Motifs in Cancer-Related Proteins, Gain Insight into CRM1-Mediated NES Export, and Search for NES-Harboring Micropeptides

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    The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the “NES-binding groove” of CRM1 was studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harboring human micropeptides.This work was supported by grants from the Spanish Government MINECO-FEDER (SAF2014-57743-R), the Basque Country Government (IT1257-19) and the University of the Basque Country (UFI11/20), as well as a fellowship from the Basque Country Government (to M.S.)

    Búsqueda de señales de transporte nucleocitoplásmico en la proteína KMT2D

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    [ES] KMT2D es una metiltransferasa que se encarga de transferir un grupo metilo a la histona H3, lo que está relacionado con la activación génica. Esta proteína es esencial en el desarrollo embrionario temprano y, además, se expresa también en numerosos tejidos adultos. Las mutaciones en el gen que codifica para la síntesis de KMT2D en la línea germinal están relacionadas con el síndrome de Kabuki, una enfermedad congénita rara. También se ha visto que las mutaciones somáticas están relacionadas con la formación de tumores, siendo KMT2D una de las proteínas más frecuentemente mutadas en el cáncer. Por su actividad metiltransferasa de histonas, se sabe que KMT2D desarrolla su función en el núcleo. Partiendo de la hipótesis inicial de que KMT2D puede tener señales de transporte nucleocitoplásmico funcionales, se realizaron análisis bioinformáticos para predecir tanto posibles señales de localización nuclear (NLS) como señales de exportación nuclear (NES) en su secuencia. Los ensayos experimentales revelaron que KMT2D tiene una NLS funcional y una NES débil. De este modo, si se identifican mutaciones relacionadas con patologías en las regiones en las que se encuentran estas señales, se podría investigar la relación del transporte alterado de KMT2D con las diferentes patologías

    Selinexor, a novel selective inhibitor of nuclear export, reduces SARS-CoV-2 infection and protects the respiratory system in vivo

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    The novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the recent global pandemic. The nuclear export protein (XPO1) has a direct role in the export of SARS-CoV proteins including ORF3b, ORF9b, and nucleocapsid. Inhibition of XPO1 induces anti-inflammatory, anti-viral, and antioxidant pathways. Selinexor is an FDA-approved XPO1 inhibitor. Through bioinformatics analysis, we predicted nuclear export sequences in the ACE-2 protein and confirmed by in vitro testing that inhibition of XPO1 with selinexor induces nuclear localization of ACE-2. Administration of selinexor inhibited viral infection prophylactically as well as therapeutically in vitro. In a ferret model of COVID-19, selinexor treatment reduced viral load in the lungs and protected against tissue damage in the nasal turbinates and lungs in vivo. Our studies demonstrated that selinexor downregulated the pro-inflammatory cytokines IL-1β, IL-6, IL-10, IFN-γ, TNF-α, and GMCSF, commonly associated with the cytokine storm observed in COVID-19 patients. Our findings indicate that nuclear export is critical for SARS-CoV-2 infection and for COVID-19 pathology and suggest that inhibition of XPO1 by selinexor could be a viable anti-viral treatment option

    Identification of nucleocytoplasmic transport signals in Rps15 and Ltv1, two proteins related to small ribosomal subunit export

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    [EN] Ribosomes are indispensable organelles for cell survival. Eukaryotic ribosomes are made up of two subunits (60S and 40S), which are independently exported from the nucleolus to the cytoplasm, where they are finally assembled to form the functional ribosome. Due to their large size, ribosomal subunits need nuclear transport receptors, or karyopherins (such as the export receptor XPO1), to move through the nuclear pore complex. Karyopherins recognize cargo proteins bearing nucleocytoplasmic transport signals (nuclear localization signals (NLSs) or nuclear export signals (NESs)). In addition to karyopherins, nucleocytoplasmic transport of the small ribosomal subunit (40S), requires other factors, such as the ribosomal protein Rps15 and the non-ribosomal protein Ltv1. Due to the role of these proteins in the export of small ribosomal subunit, we hypothesized that they could carry still uncharacterized nucleocytoplasmic transport signals. Here we describe the identification of three new functional nuclear transport signals: one NLS and one NES in Rps15, as well as one NLS in Ltv1. Our results provide new information on the specific amino acid sequences that contribute to the function of Ltv1 and Rps15 as adapters in the nuclear export of the small ribosomal subunit.[ES] Los ribosomas son orgánulos indispensables para la supervivencia de la célula. Los ribosomas eucariotas están compuestos por dos subunidades (60S y 40S), que se exportan desde el nucleolo hasta el citoplasma de forma independiente, donde son finalmente ensambladas para dar lugar al ribosoma funcional. Debido a su gran tamaño, las subunidades ribosómicas necesitan receptores de transporte nuclear, o carioferinas (como el receptor de exportación XPO1), para atravesar el complejo del poro nuclear. Las carioferinas reconocen proteínas “cargo” que portan señales de transporte nucleocitoplásmico (señales de localización nuclear (NLSs) o señales de exportación nuclear (NESs)). Además de las carioferinas, el transporte nucleocitoplásmico de la subunidad ribosómica pequeña (40S), requiere otros factores, como la proteína ribosómica Rps15 y la proteína no ribosómica Ltv1. Debido al importante papel que juegan en la exportación de la subunidad ribosómica pequeña, planteamos la hipótesis de que estas proteínas podrían poseer señales de transporte nucleocitoplásmico aún sin caracterizar. Aquí describimos la identificación de tres nuevas señales de transporte nuclear funcionales: una NLS y una NES en Rps15, así como una NLS en Ltv1. Nuestros resultados aportan nueva información sobre las secuencias de aminoácidos concretas que contribuyen al funcionamiento de Ltv1 y de Rps15 como adaptadores en la exportación nuclear de la subunidad ribosómica pequeña

    A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods

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    The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds, and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation may control its homeostasis and regulation of protein turnover is separable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final 2 positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCE The interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon-stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell and interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity

    A conserved motif in three viral movement proteins from different genera is required for host factor recruitment and cell-to-cell movement

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    [EN] Due to their minimal genomes, plant viruses are forced to hijack specific cellular pathways to ensure host colonization, a condition that most frequently involves physical interaction between viral and host proteins. Among putative viral interactors are the movement proteins, responsible for plasmodesma gating and genome binding during viral transport. Two of them, DGBp1 and DGBp2, are required for alpha-, beta- and gammacarmovirus cell-to-cell movement, but the number of DGBp-host interactors identified at present is limited. By using two different approaches, yeast two-hybrid and bimolecular fluorescence complementation assays, we found three Arabidopsis factors, eIF3g1, RPP3A and WRKY36, interacting with DGBp1s from each genus mentioned above. eIF3g1 and RPP3A are mainly involved in protein translation initiation and elongation phases, respectively, while WRKY36 belongs to WRKY transcription factor family, important regulators of many defence responses. These host proteins are not expected to be associated with viral movement, but knocking out WRKY36 or silencing either RPP3A or eIF3g1 negatively affected Arabidopsis infection by Turnip crinkle virus. A highly conserved FNF motif at DGBp1 C-terminus was required for protein-protein interaction and cell-to-cell movement, suggesting an important biological role.We thank Dr. Anne Simon and Dr. Steve A. Lommel for providing an infectious cDNA clone of the Turnip crinkle virus strain M (TCV-M) and PZP-TCV-sGFP plasmid, respectively. This work was funded by grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). J.A.N. and M.S.-S. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Ciencia, Innovacion y Universidades of Spain.Navarro Bohigues, JA.; Serra-Soriano, M.; Corachán Valencia, L.; Pallás Benet, V. (2020). 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