RESUMO
Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R-MyD88-IRAK2-PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity.
Assuntos
Adipócitos/imunologia , Inflamação/imunologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-1/metabolismo , Membranas Mitocondriais/metabolismo , Obesidade/imunologia , Adipócitos/patologia , Animais , Células Cultivadas , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Masculino , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fosforilação Oxidativa , Proibitinas , Transporte Proteico , Receptores de Interleucina-1/metabolismo , Transdução de SinaisRESUMO
Mechanisms that degrade inflammatory mRNAs are well known; however, stabilizing mechanisms are poorly understood. Here, we show that Act1, an interleukin-17 (IL-17)-receptor-complex adaptor, binds and stabilizes mRNAs encoding key inflammatory proteins. The Act1 SEFIR domain binds a stem-loop structure, the SEFIR-binding element (SBE), in the 3' untranslated region (UTR) of Cxcl1 mRNA, encoding an inflammatory chemokine. mRNA-bound Act1 directs formation of three compartmentally distinct RNA-protein complexes (RNPs) that regulate three disparate events in inflammatory-mRNA metabolism: preventing mRNA decay in the nucleus, inhibiting mRNA decapping in P bodies and promoting translation. SBE RNA aptamers decreased IL-17-mediated mRNA stabilization in vitro, IL-17-induced skin inflammation and airway inflammation in a mouse asthma model, thus providing a therapeutic strategy for autoimmune diseases. These results reveal a network in which Act1 assembles RNPs on the 3' UTRs of select mRNAs and consequently controls receptor-mediated mRNA stabilization and translation during inflammation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Inflamação/imunologia , Interleucina-17/metabolismo , Estabilidade de RNA/fisiologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Regulação da Expressão Gênica/imunologia , Inflamação/metabolismo , Interleucina-17/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Receptores de Interleucina-17/metabolismoRESUMO
S-nitrosylation is a ubiquitous protein modification emerging as a principal mechanism of nitric oxide (NO)-mediated signal transduction and cell function. S-nitrosylases can use NO synthase (NOS)-derived NO to modify selected cysteines in target proteins. Despite proteomic identification of over a thousand S-nitrosylated proteins, few S-nitrosylases have been identified. Moreover, mechanisms underlying site-selective S-nitrosylation and the potential role of specific sequence motifs remain largely unknown. Here, we describe a stimulus-inducible, heterotrimeric S-nitrosylase complex consisting of inducible NOS (iNOS), S100A8, and S100A9. S100A9 exhibits transnitrosylase activity, shuttling NO from iNOS to the target protein, whereas S100A8 and S100A9 coordinately direct site selection. A family of proteins S-nitrosylated by iNOS-S100A8/A9 were revealed by proteomic analysis. A conserved I/L-X-C-X2-D/E motif was necessary and sufficient for iNOS-S100A8/A9-mediated S-nitrosylation. These results reveal an elusive parallel between protein S-nitrosylation and phosphorylation, namely, stimulus-dependent posttranslational modification of selected targets by primary sequence motif recognition.
Assuntos
Complexos Multiproteicos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Interferon gama/metabolismo , Lipoproteínas LDL/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.
Assuntos
Células Endoteliais/metabolismo , Biossíntese de Proteínas , Fator A de Crescimento do Endotélio Vascular/genética , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Aorta/citologia , Sequência de Bases , Bovinos , Linhagem Celular , Códon de Terminação , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Alinhamento de SequênciaRESUMO
Interleukin 1ß (IL-1ß) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1ß during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1ß production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1ß, whereas ATP stimulation triggered T cell production of IL-1ß via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1ß. Together these data reveal a critical role for IL-1ß produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T/imunologia , Células Th17/imunologia , Trifosfato de Adenosina/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Caspase 8/genética , Caspase 8/imunologia , Caspase 8/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Citometria de Fluxo , Expressão Gênica/imunologia , Immunoblotting , Inflamassomos/genética , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein (MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (Eprs+/-) mice showed enhanced viremia and inflammation and delayed viral clearance. This stimulus-inducible activation of MAVS by EPRS suggests an unexpected role for the MSC as a regulator of immune responses to viral infection.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Resistência à Doença/imunologia , Interações Hospedeiro-Patógeno/imunologia , Viroses/imunologia , Viroses/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Animais , Antivirais/farmacologia , Modelos Animais de Doenças , Imunidade Inata , Camundongos , Camundongos Knockout , Peptídeos/farmacologia , Fosforilação , Ligação Proteica , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/virologia , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/imunologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Ubiquitinação , Viroses/virologia , Replicação ViralRESUMO
Posttranscriptional regulatory mechanisms superimpose "fine-tuning" control upon "on-off" switches characteristic of gene transcription. We have exploited computational modeling with experimental validation to resolve an anomalous relationship between mRNA expression and protein synthesis. The GAIT (gamma-interferon-activated inhibitor of translation) complex repressed VEGF-A synthesis to a low, constant rate independent of VEGF-A mRNA expression levels. Dynamic model simulations predicted an inhibitory GAIT-element-interacting factor to account for this relationship and led to the identification of a truncated form of glutamyl-prolyl tRNA synthetase (EPRS), a GAIT constituent that mediates binding to target transcripts. The truncated protein, EPRS(N1), shields GAIT-element-bearing transcripts from the inhibitory GAIT complex, thereby dictating a "translational trickle" of GAIT target proteins. EPRS(N1) mRNA is generated by polyadenylation-directed conversion of a Tyr codon in the EPRS-coding sequence to a stop codon (PAY(∗)). Genome-wide analysis revealed multiple candidate PAY(∗) targets, including the authenticated target RRM1, suggesting a general mechanism for production of C terminus-truncated regulatory proteins.
Assuntos
Aminoacil-tRNA Sintetases/genética , Regulação da Expressão Gênica , Genoma Humano , Biossíntese de Proteínas , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Códon de Terminação , Humanos , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Poliadenilação , Transcriptoma , Células U937 , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Multisite phosphorylation of kinases can induce on-off or graded regulation of catalytic activity; however, its influence on substrate specificity remains unclear. Here, we show that multisite phosphorylation of ribosomal protein S6 kinase 1 (S6K1) alters target selection. Agonist-inducible phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) by S6K1 in monocytes and adipocytes requires not only canonical phosphorylation at Thr389 by mTORC1 but also phosphorylation at Ser424 and Ser429 in the C terminus by cyclin-dependent kinase 5 (Cdk5). S6K1 phosphorylation at these additional sites induces a conformational switch and is essential for high-affinity binding and phosphorylation of EPRS, but not canonical S6K1 targets, e.g., ribosomal protein S6. Unbiased proteomic analysis identified additional targets phosphorylated by multisite phosphorylated S6K1 in insulin-stimulated adipocytes-namely, coenzyme A synthase, lipocalin 2, and cortactin. Thus, embedded within S6K1 is a target-selective kinase phospho-code that integrates signals from mTORC1 and Cdk5 to direct an insulin-stimulated, post-translational metabolon determining adipocyte lipid metabolism.
Assuntos
Adipócitos/enzimologia , Metabolismo dos Lipídeos , Células Mieloides/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Aminoacil-tRNA Sintetases/metabolismo , Animais , Quinase 5 Dependente de Ciclina/metabolismo , Ativação Enzimática , Células HEK293 , Células Hep G2 , Humanos , Insulina/farmacologia , Interferon gama/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Células Mieloides/efeitos dos fármacos , Fosforilação , Proteômica/métodos , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais , Especificidade por Substrato , Células U937RESUMO
Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.
Assuntos
Neoplasias da Mama , Núcleo Celular , Poli(ADP-Ribose) Polimerase-1 , Proteínas Proto-Oncogênicas c-akt , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Núcleo Celular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Linhagem Celular Tumoral , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/genética , Transporte Ativo do Núcleo Celular , Sinais de Localização Nuclear/metabolismoRESUMO
In the developing nervous system, neural stem cells (NSCs) use temporal patterning to generate a wide variety of different neuronal subtypes. In Drosophila, the temporal transcription factors, Hunchback, Kruppel, Pdm and Castor, are sequentially expressed by NSCs to regulate temporal identity during neurogenesis. Here, we identify a new temporal transcription factor that regulates the transition from the Pdm to Castor temporal windows. This factor, which we call Chronophage (or 'time-eater'), is homologous to mammalian CTIP1 (Bcl11a) and CTIP2 (Bcl11b). We show that Chronophage binds upstream of the castor gene and regulates its expression. Consistent with Chronophage promoting a temporal switch, chronophage mutants generate an excess of Pdm-specified neurons and are delayed in generating neurons associated with the Castor temporal window. In addition to promoting the Pdm to Castor transition, Chronophage also represses the production of neurons generated during the earlier Hunchback and Kruppel temporal windows. Genetic interactions with Hunchback and Kruppel indicate that Chronophage regulates NSC competence to generate Hunchback- and Kruppel-specified neurons. Taken together, our results suggest that Chronophage has a conserved role in temporal patterning and neuronal subtype specification.
Assuntos
Proteínas de Drosophila , Células-Tronco Neurais , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mamíferos/metabolismo , Células-Tronco Neurais/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Complexes of two or more proteins form many, if not most, of the intracellular "machines" that execute physical and chemical work, and transmit information. Complexes can form from stochastic post-translational interactions of fully formed proteins, but recent attention has shifted to co-translational interactions in which the most common mechanism involves binding of a mature constituent to an incomplete polypeptide emerging from a translating ribosome. Studies in yeast have revealed co-translational interactions during formation of multiple major complexes, and together with recent mammalian cell studies, suggest widespread utilization of the mechanism. These translation-dependent interactions can involve a single or multiple mRNA templates, can be uni- or bi-directional, and can use multi-protein sub-complexes as a binding component. Here, we discuss benefits of co-translational complex assembly including accuracy and efficiency, overcoming hidden interfaces, localized and hierarchical assembly, and reduction of orphan protein degradation, toxicity, and dominant-negative pathogenesis, all serving to improve cell fitness.
Assuntos
Biossíntese de Proteínas , Ribossomos , Animais , Ribossomos/metabolismo , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mamíferos/genéticaRESUMO
Amino acid ligation to cognate transfer RNAs (tRNAs) is catalyzed by aminoacyl-tRNA synthetases (aaRSs)-essential interpreters of the genetic code during translation. Mammalian cells harbor 20 cytoplasmic aaRSs, out of which 9 (in 8 proteins), with 3 non-aaRS proteins, AIMPs 1 to 3, form the â¼1.25-MDa multi-tRNA synthetase complex (MSC). The function of MSC remains uncertain, as does its mechanism of assembly. Constituents of multiprotein complexes encounter obstacles during assembly, including inappropriate interactions, topological constraints, premature degradation of unassembled subunits, and suboptimal stoichiometry. To facilitate orderly and efficient complex formation, some complexes are assembled cotranslationally by a mechanism in which a fully formed, mature protein binds a nascent partner as it emerges from the translating ribosome. Here, we show out of the 121 possible interaction events between the 11 MSC constituents, 15 are cotranslational. AIMPs are involved in the majority of these cotranslational interactions, suggesting they are not only critical for MSC structure but also for assembly. Unexpectedly, several cotranslational events involve more than the usual dyad of interacting proteins. We show two modes of cotranslational interaction, namely a "multisite" mechanism in which two or more mature proteins bind the same nascent peptide at distinct sites and a second "piggy-back" mechanism in which a mature protein carries a second fully formed protein and binds to a single site on an emerging peptide. Multimodal mechanisms of cotranslational interaction offer a diversity of pathways for ordered, piecewise assembly of small subcomplexes into larger heteromultimeric complexes such as the mammalian MSC.
Assuntos
Aminoacil-tRNA Sintetases , Aminoacil-tRNA Sintetases/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Ribossomos/metabolismoRESUMO
Multiprotein assemblages are the intracellular workhorses of many physiological processes. Assembly of constituents into complexes can be driven by stochastic, domain-dependent, posttranslational events in which mature, folded proteins specifically interact. However, inaccessibility of interacting surfaces in mature proteins (e.g., due to "buried" domains) can obstruct complex formation. Mechanisms by which multiprotein complex constituents overcome topological impediments remain enigmatic. For example, the heterodimeric complex formed by EBP50 and ezrin must address this issue as the EBP50-interacting domain in ezrin is obstructed by a self-interaction that occupies the EBP50 binding site. Here, we show that the EBP50-ezrin complex is formed by a cotranslational mechanism in which the C terminus of mature, fully formed EBP50 binds the emerging, ribosome-bound N-terminal FERM domain of ezrin during EZR mRNA translation. Consistent with this observation, a C-terminal EBP50 peptide mimetic reduces the cotranslational interaction and abrogates EBP50-ezrin complex formation. Phosphorylation of EBP50 at Ser339 and Ser340 abrogates the cotranslational interaction and inhibits complex formation. In summary, we show that the function of eukaryotic mRNA translation extends beyond "simple" generation of a linear peptide chain that folds into a tertiary structure, potentially for subsequent complex assembly; importantly, translation can facilitate interactions with sterically inaccessible domains to form functional multiprotein complexes.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sítios de Ligação , Sistemas CRISPR-Cas , Clonagem Molecular , Proteínas do Citoesqueleto/genética , DNA Complementar , Regulação da Expressão Gênica , Inativação Gênica , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Modelos Moleculares , Fosfoproteínas/genética , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
BACKGROUND: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers. METHODS: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing. RESULTS: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2. CONCLUSIONS: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.
Assuntos
Antígenos CD55 , Núcleo Celular , Cromatina , Cisplatino , Resistencia a Medicamentos Antineoplásicos , Histonas , Células-Tronco Neoplásicas , Neoplasias Ovarianas , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , Feminino , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Camundongos , Antígenos CD55/metabolismo , Antígenos CD55/genética , Linhagem Celular Tumoral , Histonas/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Metilação , Ensaios Antitumorais Modelo de Xenoenxerto , Antineoplásicos/farmacologia , Transporte ProteicoRESUMO
Many tissues contain multipotent stem cells that are critical for maintaining tissue function. In Caenorhabditis elegans, germline stem cells allow gamete production to continue in adulthood. In the gonad, GLP-1/Notch signaling from the distal tip cell niche to neighboring germ cells activates a complex regulatory network to maintain a stem cell population. GLP-1/Notch signaling positively regulates production of LST-1 and SYGL-1 proteins that, in turn, interact with a set of PUF/FBF proteins to positively regulate the stem cell fate. We previously described sog (suppressor of glp-1 loss of function) and teg (tumorous enhancer of glp-1 gain of function) genes that limit the stem cell fate and/or promote the meiotic fate. Here, we show that sog-10 is allelic to nhl-2. NHL-2 is a member of the conserved TRIM-NHL protein family whose members can bind RNA and ubiquitinate protein substrates. We show that NHL-2 acts, at least in part, by inhibiting the expression of PUF-3 and PUF-11 translational repressor proteins that promote the stem cell fate. Two other negative regulators of stem cell fate, CGH-1 (conserved germline helicase) and ALG-5 (Argonaute protein), may work with NHL-2 to modulate the stem cell population. In addition, NHL-2 activity promotes the male germ cell fate in XX animals.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Células Germinativas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Masculino , RNA/metabolismo , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismoRESUMO
Translational readthrough generates proteins with extended C-termini, which often possess distinct properties. Here, we have used various reporter assays to demonstrate translational readthrough of AGO1 mRNA. Analysis of ribosome profiling data and mass spectrometry data provided additional evidence for translational readthrough of AGO1. The endogenous readthrough product, Ago1x, could be detected by a specific antibody both in vitro and in vivo. This readthrough process is directed by a cis sequence downstream of the canonical AGO1 stop codon, which is sufficient to drive readthrough even in a heterologous context. This cis sequence has a let-7a miRNA-binding site, and readthrough is promoted by let-7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post-transcriptional gene silencing, due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1.
Assuntos
Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , MicroRNAs/genética , Biossíntese de Proteínas , Proteínas Argonautas/química , Autoantígenos/metabolismo , Sítios de Ligação , Códon de Terminação , Fatores de Iniciação em Eucariotos/química , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transdução de SinaisRESUMO
Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that serve a foundational role in the efficient and accurate translation of genetic information from messenger RNA to proteins. These proteins play critical, non-canonical functions in a multitude of cellular processes. Multiple viruses are known to hijack the functions of aaRSs for proviral outcomes, while cells modify antiviral responses through non-canonical functions of certain synthetases. Recent findings have revealed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronaviral disease 19 (COVID-19), utilizes canonical and non-canonical functions of aaRSs, establishing a complex interplay of viral proteins, cellular factors and host aaRSs. In a striking example, an unconventional multi-aaRS complex consisting of glutamyl-prolyl-, lysyl-, arginyl- and methionyl-tRNA synthetases interact with a previously unknown RNA-element in the 3'-end of SARS-CoV-2 genomic and subgenomic RNAs. This review aims to highlight the aaRS-SARS-CoV-2 interactions identified to date, with possible implications for the biology of host aaRSs in SARS-CoV-2 infection.
Assuntos
Aminoacil-tRNA Sintetases , COVID-19 , Humanos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , SARS-CoV-2/genética , Genoma , RNA de Transferência/metabolismoRESUMO
Adiponectin, an adipocyte-specific secretory protein encoded by the ADIPOQ gene has a causal role in insulin resistance. Anti-diabetic drugs increase plasma adiponectin by a poorly understood, post-transcriptional mechanism enhancing insulin sensitivity. Deletion analysis of a reporter bearing the mouse Adipoq mRNA 5'-leader identified an inhibitory cis-regulatory sequence. The 5'-leader harbours two potential upstream open reading frames (uORFs) overlapping the principal downstream ORF. Mutation of the uORF ATGs increased reporter translation ~3-fold, indicative of a functional uORF. uORFs are common in mammalian mRNAs; however, only a select group resist translational repression by the integrated stress response (ISR). Thapsigargin (TG), which induces endoplasmic reticulum (ER) stress and the ISR, enhanced expression of a reporter bearing the Adipoq 5'-leader; polysome profiling verified translation-stimulation. TG-stimulated translation was absent in cells defective in Ser51 phosphorylation of eukaryotic initiation factor 2α (eIF2α), required for the ISR. To determine its role in expression and function of endogenous adiponectin, the upstream uORF was disrupted by CRISPR-Cas9-mediated mutagenesis of differentiated mouse 3T3-L1 adipocytes. uORF disruption in adipocytes increased adiponectin expression, triacylglycerol accumulation, and glucose uptake, and inhibited paracrine muscle and liver cell expression of gluconeogenic enzymes, establishing an important role of the uORF in adiponectin-mediated responses to stress.
Assuntos
Adipócitos , Adiponectina , Animais , Camundongos , Adiponectina/genética , Fases de Leitura Aberta , Células 3T3-L1 , Transporte Biológico , MamíferosRESUMO
Metabolic pathways that contribute to adiposity and ageing are activated by the mammalian target of rapamycin complex 1 (mTORC1) and p70 ribosomal protein S6 kinase 1 (S6K1) axis. However, known mTORC1-S6K1 targets do not account for observed loss-of-function phenotypes, suggesting that there are additional downstream effectors of this pathway. Here we identify glutamyl-prolyl-tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes to adiposity and ageing. Phosphorylation of EPRS at Ser999 by mTORC1-S6K1 induces its release from the aminoacyl tRNA multisynthetase complex, which is required for execution of noncanonical functions of EPRS beyond protein synthesis. To investigate the physiological function of EPRS phosphorylation, we generated Eprs knock-in mice bearing phospho-deficient Ser999-to-Ala (S999A) and phospho-mimetic (S999D) mutations. Homozygous S999A mice exhibited low body weight, reduced adipose tissue mass, and increased lifespan, similar to S6K1-deficient mice and mice with adipocyte-specific deficiency of raptor, an mTORC1 constituent. Substitution of the EprsS999D allele in S6K1-deficient mice normalized body mass and adiposity, indicating that EPRS phosphorylation mediates S6K1-dependent metabolic responses. In adipocytes, insulin stimulated S6K1-dependent EPRS phosphorylation and release from the multisynthetase complex. Interaction screening revealed that phospho-EPRS binds SLC27A1 (that is, fatty acid transport protein 1, FATP1), inducing its translocation to the plasma membrane and long-chain fatty acid uptake. Thus, EPRS and FATP1 are terminal mTORC1-S6K1 axis effectors that are critical for metabolic phenotypes.