RESUMO
Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2â»/â»Î³câ»/â» mice engrafted with either Tre-transduced primary CD4⺠T cells, or Tre-transduced CD34⺠hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV.
Assuntos
Terapia Genética/métodos , Infecções por HIV , Repetição Terminal Longa de HIV , HIV-1/metabolismo , Integrases/metabolismo , Provírus/metabolismo , Animais , Vetores Genéticos , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/terapia , HIV-1/genética , Humanos , Integrases/genética , Camundongos , Camundongos Knockout , Provírus/genética , Transdução Genética , Quimeras de Transplante , Integração Viral/genéticaRESUMO
Mature dendritic cells (DC), activated lymphocytes, mononuclear cells and neutrophils express CD83, a surface protein apparently necessary for effective DC-mediated activation of naïve T-cells and T-helper cells, thymic T-cell maturation and the regulation of B-cell activation and homeostasis. Although a defined ligand of CD83 remains elusive, the multiple cellular subsets expressing CD83, as well as its numerous potential implications in immunological processes suggest that CD83 plays an important regulatory role in the mammalian immune system. Lately, nucleocytoplasmic translocation of CD83 mRNA was shown to be mediated by direct interaction between the shuttle protein HuR and a novel post-transcriptional regulatory element (PRE) located in the CD83 transcript's coding region. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export through the CRM1 protein translocation pathway. More recently, the cellular phosphoprotein and HuR ligand ANP32B (APRIL) was demonstrated to be directly involved in this intracellular transport process by linking the CD83 mRNA:HuR ribonucleoprotein (RNP) complex with the CRM1 export receptor. Casein kinase II regulates this process by phosphorylating ANP32B. Here, we identify another RNA binding protein, AUF1 (hnRNP D) that directly interacts with CD83 PRE. Unlike HuR:PRE binding, this interaction has no impact on intracellular trafficking of CD83 mRNA-containing complexes; but it does regulate translation of CD83 mRNA. Thus, our data shed more light on the complex process of post-transcriptional regulation of CD83 expression. Interfering with this process may provide a novel strategy for inhibiting CD83, and thereby cellular immune activation.
Assuntos
Antígenos CD/genética , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Imunoglobulinas/genética , Glicoproteínas de Membrana/genética , Biossíntese de Proteínas , Animais , Antígenos CD/biossíntese , Antígenos CD/metabolismo , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , Imunoglobulinas/biossíntese , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Interferência de RNA , Sequências Reguladoras de Ácido Ribonucleico , Antígeno CD83RESUMO
Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Recombinases/metabolismo , HIV-1/fisiologia , Provírus/genética , Repetição Terminal Longa de HIV/genética , Infecções por HIV/terapia , Vetores Genéticos/genéticaRESUMO
Fully mature DC and, to a lesser extent, activated T and B cells express CD83, a surface molecule that appears to fulfil an important role in efficient T-cell activation. Recently, it has been shown that CD83 mRNA is transported from the nucleus to the cytoplasm by an uncommon route, involving the cellular RNA-binding protein HuR and the nuclear export receptor CRM1. Moreover, the shuttle phosphoprotein APRIL (ANP32B) has been shown to be required for HuR-mediated nucleocytoplasmic translocation of the CD83 mRNA by acting as an adaptor that links HuR and CRM1. Here, we are able to report that casein kinase 2 (CK2) phosphorylates APRIL on residue threonine244 (Thr(244)) and demonstrate that the CK2-specific inhibitor 4,5,6,7-tetrabromo-2-azabenzimidazole abolishes CD83 expression in activated Jurkat T cells by interfering with the nucleocytoplasmic translocation of CD83 mRNA. Depletion and knockdown studies demonstrate that the CK2 alpha' subunit is necessary for this regulation, whereas the CK2 alpha subunit seems to be dispensable. Taken together, the data presented significantly extend our knowledge of the complex regulation of CD83 mRNA processing and provides a novel strategy to interfere with CD83 expression.
Assuntos
Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Caseína Quinase II/metabolismo , Imunoglobulinas/metabolismo , Carioferinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Antígenos CD/efeitos dos fármacos , Caseína Quinase II/antagonistas & inibidores , Linhagem Celular Tumoral , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Vetores Genéticos/metabolismo , Células HeLa , Humanos , Imunoglobulinas/efeitos dos fármacos , Células Jurkat , Glicoproteínas de Membrana/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Fosforilação/fisiologia , Transfecção , Triazóis/farmacologia , Proteína Exportina 1 , Antígeno CD83RESUMO
ANP32B belongs to a family of evolutionary conserved acidic nuclear phosphoproteins (ANP32A-H). Family members have been described as multifunctional regulatory proteins and proto-oncogenic factors affecting embryonic development, cell proliferation, apoptosis, and gene expression at various levels. Involvement of ANP32B in multiple processes of cellular life is reflected by the previous finding that systemic gene knockout (KO) of Anp32b leads to embryonic lethality in mice. Here, we demonstrate that a conditional KO of Anp32b is well tolerated in adult animals. However, after immune activation splenocytes isolated from Anp32b KO mice showed a strong commitment towards Th17 immune responses. Therefore, we further analyzed the respective animals in vivo using an experimental autoimmune encephalomyelitis (EAE) model. Interestingly, an exacerbated clinical score was observed in the Anp32b KO mice. This was accompanied by the finding that animal-derived T lymphocytes were in a more activated state, and RNA sequencing analyses revealed hyperactivation of several T lymphocyte-associated immune modulatory pathways, attended by significant upregulation of Tfh cell numbers that altogether might explain the observed strong autoreactive processes. Therefore, Anp32b appears to fulfill a role in regulating adequate adaptive immune responses and, hence, may be involved in dysregulation of pathways leading to autoimmune disorders and/or immune deficiencies.
Assuntos
Imunidade Adaptativa/genética , Proteínas de Ciclo Celular/genética , Encefalomielite Autoimune Experimental/genética , Inflamação/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Imunidade Adaptativa/imunologia , Animais , Apoptose/genética , Proliferação de Células/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica/genética , Humanos , Fatores Imunológicos/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Células Th17/imunologiaRESUMO
The delivery of therapeutic genes for treatment of inherited or infectious diseases frequently requires lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSC). Optimized transduction protocols with a therapeutic goal aim to maximize the number of transduction-positive cells while limiting the vector copy number that reach each individual cell. Importantly, the transduced HSC should maintain their "stem-like" properties. Here, we analyzed LentiBOOST™ reagent, a membrane-sealing poloxamer, with respect to enhancing lentiviral transduction of CD34+ peripheral blood stem cells. We demonstrate that inclusion of LentiBOOST™ in a standard HSC transduction protocol yields high transduction efficiencies while preserving the ability of the transduced HSC to differentiate into various hematopoietic lineages. Thus, LentiBOOST™ reagent can significantly improve lentiviral CD34+ HSC transduction protocols with the potential to improve production of gene-modified cell products.
Assuntos
Vetores Genéticos/imunologia , HIV-1/genética , Células-Tronco Hematopoéticas/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Linfócitos T/imunologia , Transdução Genética/métodos , Antígenos CD34/genética , Antígenos CD34/imunologia , Diferenciação Celular , Linhagem Celular , Linhagem da Célula/imunologia , Variações do Número de Cópias de DNA , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Células HEK293 , HIV-1/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Poloxâmero/química , Cultura Primária de Células , Protaminas/química , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/normas , Linfócitos T/citologia , Linfócitos T/virologia , Transdução Genética/instrumentação , TransgenesRESUMO
In metazoans, the nuclear export of bulk mRNAs is mediated by the export receptor TAP, together with its binding partner p15. A number of viral mRNAs, including the unspliced and partially spliced mRNA species of the human immunodeficiency virus (HIV), however, use an alternative export route via the importin beta-related export receptor CRM1. This raises the question of whether a subset of cellular mRNAs might be exported by CRM1 as well. To identify such mRNAs, we performed a systematic screen in different cell lines, using representational difference analyses of cDNA (cDNA-RDA). In HeLa and Cl-4 cells no cellular transcripts could be identified as exported via CRM1. In contrast, we found a number of CRM1-dependent mRNAs in Jurkat T cells, most of which are induced during a T cell response. One of the identified gene products, the dendritic cell marker CD83, was analyzed in detail. CD83 expression depends on a functional CRM1 pathway in activated Jurkat T cells as well as in a heterologous expression system, independent of activation. Our results point to an important role of the CRM1-dependent export pathway for the expression of CD83 and other genes under conditions of T cell activation.
Assuntos
Carioferinas/metabolismo , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Linfócitos T/metabolismo , Transporte Ativo do Núcleo Celular , Antígenos CD/genética , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Expressão Gênica , Genes env , HIV/genética , Células HeLa , Humanos , Imunoglobulinas/genética , Técnicas In Vitro , Células Jurkat , Carioferinas/antagonistas & inibidores , Ativação Linfocitária/genética , Glicoproteínas de Membrana/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Linfócitos T/imunologia , Proteína Exportina 1 , Antígeno CD83RESUMO
Activation of eukaryotic translation initiation factor eIF5A requires a posttranslational modification, forming the unique amino acid hypusine. This activation is mediated by two enzymes, deoxyhypusine synthase, DHS, and deoxyhypusine hydroxylase, DOHH. The impact of this enzymatic complex on the life cycle of a fungal pathogen is unknown. Plant pathogenic ascomycetes possess a single copy of the eIF5A activated by hypusination. We evaluated the importance of imbalances in eIF5A hypusination in Fusarium graminearum, a devastating fungal pathogen of cereals. Overexpression of DHS leads to increased virulence in wheat, elevated production of the mycotoxin deoxynivalenol, more infection structures, faster wheat tissue invasion in plants and increases vegetatively produced conidia. In contrast, overexpression of DOHH completely prevents infection structure formation, pathogenicity in wheat and maize, leads to overproduction of ROS, reduced DON production and increased sexual reproduction. Simultaneous overexpression of both genes restores wild type-like phenotypes. Analysis of eIF5A posttranslational modification displayed strongly increased hypusinated eIF5A in DOHH overexpression mutant in comparison to wild type, and the DHS overexpression mutants. These are the first results pointing to different functions of differently modified eIF5A.
Assuntos
Fusarium/fisiologia , Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Fusarium/patogenicidade , Expressão Gênica , Genes Fúngicos , Lisina/biossíntese , Viabilidade Microbiana/genética , Mutação , Micotoxinas/biossíntese , Fatores de Iniciação de Peptídeos/genética , Doenças das Plantas , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/genética , Triticum/metabolismo , Triticum/microbiologia , Virulência , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.
Assuntos
Antivirais/farmacologia , Evolução Molecular Direcionada/métodos , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Provírus/efeitos dos fármacos , Recombinases/farmacologia , Integração Viral/efeitos dos fármacos , Animais , Antivirais/metabolismo , Sequência de Bases , Células Cultivadas , HIV-1/genética , Humanos , Camundongos , Dados de Sequência Molecular , Provírus/genética , Recombinases/metabolismo , Integração Viral/genéticaRESUMO
Current antiviral therapies cannot cure hepatitis B virus (HBV) infection; successful HBV eradication would require inactivation of the viral genome, which primarily persists in host cells as episomal covalently closed circular DNA (cccDNA) and, to a lesser extent, as chromosomally integrated sequences. However, novel designer enzymes, such as the CRISPR/Cas9 RNA-guided nuclease system, provide technologies for developing advanced therapy strategies that could directly attack the HBV genome. For therapeutic application in humans, such designer nucleases should recognize various HBV genotypes and cause minimal off-target effects. Here, we identified cross-genotype conserved HBV sequences in the S and X region of the HBV genome that were targeted for specific and effective cleavage by a Cas9 nickase. This approach disrupted not only episomal cccDNA and chromosomally integrated HBV target sites in reporter cell lines, but also HBV replication in chronically and de novo infected hepatoma cell lines. Our data demonstrate the feasibility of using the CRISPR/Cas9 nickase system for novel therapy strategies aiming to cure HBV infection.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Desoxirribonuclease I/genética , Genoma Viral/genética , Vírus da Hepatite B/genética , Hepatite B/genética , Fases de Leitura Aberta/genética , Inativação GênicaRESUMO
The major obstacle to cure infections with human immunodeficiency virus (HIV-1) is integrated proviral genomes, which are not eliminated by antiretroviral therapies (ART). Treatment approaches with latency-reversing agents (LRAs) aim at inducing provirus expression to tag latently-infected cells for clearance through viral cytopathic effects or cytotoxic T cell (CTL) responses. However, the currently tested LRAs reveal evident drawbacks as gene expression is globally induced and viral outgrowth is insecure. Here, we present transcription activator-like effector (TALE) proteins as potent tools to activate HIV-1 specifically. The large variety of circulating HIV-1 strains and, accordingly, integrated proviruses was addressed by the programmable DNA-specificity of TALEs. Using customized engineered TALEs, a substantial transcription activation and viral outgrowth was achieved with cells obtained from different HIV-1 patients. Our data suggest that TALEs may be useful tools in future strategies aimed at removing HIV-1 reservoirs.
Assuntos
Infecções por HIV/metabolismo , HIV-1/fisiologia , Fatores de Transcrição/metabolismo , Ativação Viral , Regulação Viral da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Humanos , Família Multigênica , Especificidade da Espécie , Fatores de Transcrição/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Latência ViralRESUMO
Current drugs against HIV can suppress the progression to AIDS but cannot clear the patient from the virus. Because of potential side effects of these drugs and the possible development of drug resistance, finding a cure for HIV infection remains a high priority of HIV/AIDS research. We recently generated a recombinase (termed Tre) tailored to efficiently eradicate the provirus from the host genome of HIV-1 infected cells by specifically targeting a sequence that is present in the long terminal repeats (LTRs) of the viral DNA [1]. In vivo analyses in HIV-infected humanized mice demonstrated highly significant antiviral effects of Tre recombinase [2]. However, the fact that Tre recognizes a particular HIV-1 subtype A strain may limit its broad therapeutic application. To advance our Tre-based strategy towards a universally efficient cure, we have engineered a new, universal recombinase (uTre) applicable to the majority of HIV-1 infections by the various virus strains and subtypes. We employed the search tool SeLOX [3] in order to find a well-conserved HIV-1 proviral sequence that could serve as target site for a universal Tre from sequences compiled in the Los Alamos HIV Sequence Database. We selected a candidate (termed loxLTRu) with a mean conservation rate of 94% throughout the major HIV-1 subtype groups A, B and C. We applied loxLTRu as substrate in our established substrate-linked protein evolution (SLiPE) process [4] and evolved the uTre recombinase in 142 evolution cycles. Highly specific enzymatic activity on loxLTRu is demonstrated for uTre in both Escherichia coli and human cells. Naturally occurring viral variants with single mutations within the loxLTRu sequence are also shown to be efficiently targeted by uTre, further increasing the range of applicability of the recombinase. Potential off-target sites in the human genome are not recombined by uTre. Furthermore, uTre expression in primary human T cells shows no obvious Tre-related cytopathic or genotoxic effects. Finally, uTre expressing mice show no undesired phenotypes during their normal lifespan. We have developed a broad-range HIV-1 LTR specific recombinase that has the potential to be effective against the vast majority of HIV-1 strains and to cure HIV-1 infected cells from the infection. These results strongly encouraged us in our confidence that a Tre recombinase-mediated HIV eradication strategy may become a valuable component of a future therapy for HIV-infected patients.
RESUMO
Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1-3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein:RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway.
Assuntos
ADP-Ribosil Ciclase 1/genética , Proteínas ELAV/genética , RNA Mensageiro/genética , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Primers do DNA , Humanos , Ativação Linfocitária , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologiaRESUMO
Dendritic cells (DC) are the most potent antigen-presenting cells (APC) of the immune system and are specialized to activate T as well as B cell-dependent immune responses. Mature DC are characterized by expression of CD83, a surface molecule that has been postulated to be required for efficient DC activity. Here we show that Leptomycin B (LMB), a highly specific inhibitor of the nuclear export receptor CRM1, abrogates the ability of DC to stimulate T cells in an allogeneic mixed lymphocyte reaction. Interestingly, this effect correlates with down-regulation of CD83, CD80 and CD86 surface expression during DC maturation, whereas other investigated DC surface molecules, such as MHC class I and II molecules are not significantly affected. Analysis of RNA distribution reveals that particularly the stimulated expression of CD83 depended on a functional CRM1 export receptor. Taken together, the presented data show a critical involvement of the CRM1 transport receptor in DC maturation, most likely by enabling efficient nucleo-cytoplasmic translocation of specific mRNAs. Thus, interference with this pathway may provide new strategies to modulate DC function and, subsequently, DC-mediated immune responses.
Assuntos
Células Dendríticas/imunologia , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Antígenos CD/biossíntese , Diferenciação Celular , Células Dendríticas/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Humanos , Imunoglobulinas/biossíntese , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/biossíntese , Neutrófilos/imunologia , Proteína Exportina 1 , Antígeno CD83RESUMO
Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system and are able to sensitize even naïve T cells. Mature DC are characterized by expression of CD83, a surface molecule that is proposed to be involved in efficient T cell activation. It has been recently shown that CD83 mRNA is transported from the nucleus to the cytoplasm in a HuR- and CRM1-dependent manner. Therefore we here investigated the impact of two known protein ligands of HuR, pp32 and APRIL, on CD83 expression. Both pp32 (ANP32A) and APRIL (ANP32B) are shuttle proteins, and it has been reported earlier that these HuR ligands can act as adaptors that link HuR and the CRM1-specific nuclear export pathway. By employing RNA interference (RNAi) technology we demonstrate that pp32 is dispensable for CD83 expression, whereas APRIL contributes to the nuclear export and subsequent translation of CD83 mRNA. Furthermore, we have determined the nuclear import signal (NLS) as well as the nuclear export signal (NES) of human APRIL. Moreover, we analyzed the status of phosphorylation of endogenous APRIL and identified threonine 244 to be an as yet unrecognized phosphate acceptor. Finally, we were able to show that phosphorylation of this specific amino acid residue regulates the nuclear export of APRIL. In sum, we report here the signal sequences in APRIL that mediate its intracellular trafficking and provide evidence that this protein ligand of HuR is an important player in the post-transcriptional regulation of CD83 expression by affecting the nucleocytoplasmic translocation of CD83 mRNA.
Assuntos
Antígenos CD/biossíntese , Antígenos de Superfície/metabolismo , Núcleo Celular/metabolismo , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/fisiologia , Imunoglobulinas/biossíntese , Glicoproteínas de Membrana/biossíntese , Proteínas de Ligação a RNA/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Apresentação de Antígeno/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Células COS , Núcleo Celular/genética , Núcleo Celular/imunologia , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/imunologia , Citoplasma/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Células HeLa , Humanos , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Ligantes , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Antígeno CD83RESUMO
Dendritic cells are the most potent of the antigen-presenting cells and are characterized by surface expression of CD83. Here, we show that the coding region of CD83 mRNA contains a novel cis-acting structured RNA element that binds to HuR, a member of the ELAV family of AU-rich element RNA-binding proteins. Transient transfection of mammalian cells demonstrated that this CD83 mRNA-derived element acts as a post-transcriptional regulatory element in cells overexpressing HuR. Notably, binding of HuR to the CD83 post-transcriptional regulatory element did not affect mRNA stability. Using RNA interference, we show that HuR mediated efficient expression of CD83. In particular, HuR was required for cytoplasmic accumulation of CD83 transcripts. Likewise, inhibition of the CRM1 nuclear export pathway by leptomycin B or overexpression of a defective form of the nucleoporin Nup214/CAN diminished cytoplasmic CD83 mRNA levels. In summary, the data presented demonstrate that the HuR-CRM1 axis affects the nucleocytoplasmic translocation of CD83 mRNA under regular physiological conditions.
Assuntos
Antígenos CD/biossíntese , Antígenos de Superfície/fisiologia , Regulação da Expressão Gênica , Imunoglobulinas/biossíntese , Carioferinas/fisiologia , Glicoproteínas de Membrana/biossíntese , Proteínas de Ligação a RNA/fisiologia , RNA/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ácidos Graxos Insaturados/farmacologia , Inativação Gênica , Genes Reporter , Vetores Genéticos , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Células Jurkat , Cinética , Luciferases/metabolismo , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Reação em Cadeia da Polimerase , Ligação Proteica , Biossíntese de Proteínas , Transporte Proteico , RNA/química , RNA/metabolismo , Interferência de RNA , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteína Exportina 1 , Antígeno CD83RESUMO
The archetypal human tumor suppressor p53 is considered to have unique transactivation properties. The assumption is based on the fact that additionally identified human p53 isoforms lack transcriptional activity. However, we provide evidence for the existence of an alternatively spliced p53 isoform (Deltap53) that exerts its transcriptional activity independent from p53. In contrast to p53, Deltap53 transactivates the endogenous p21 and 14-3-3sigma but not the mdm2, bax, and PIG3 promoter. Cell cycle studies showed that Deltap53 displays its differential transcriptional activity only in damaged S phase cells. Upon activation of the ATR-intra-S phase checkpoint, Deltap53, but not p53, transactivates the Cdk inhibitor p21. Induction of p21 results in downregulation of cyclin A-Cdk activity and accordingly attenuation of S phase progression. Data demonstrate that the Deltap53-p21-cyclin A-Cdk pathway is crucial to facilitate uncoupling of repair and replication events, indicating that Deltap53 is an essential element of the ATR-intra-S phase checkpoint.