RESUMO
GCN2 is a conserved receptor kinase activating the Integrated Stress Response (ISR) in eukaryotic cells. The ISR kinases detect accumulation of stress molecules and reprogram translation from basal tasks to preferred production of cytoprotective proteins. GCN2 stands out evolutionarily among all protein kinases due to the presence of a h istidyl t R NA s ynthetase-like (HRSL) domain, which arises only in GCN2 and is located next to the kinase domain. How HRSL contributes to GCN2 signaling remains unknown. Here we report a 3.2 Å cryo-EM structure of HRSL from thermotolerant yeast Kluyveromyces marxianus . This structure shows a constitutive symmetrical homodimer featuring a compact helical-bundle structure at the junction between HRSL and kinase domains, in the core of the receptor. Mutagenesis demonstrates that this junction structure activates GCN2 and indicates that our cryo-EM structure captures the active signaling state of HRSL. Based on these results, we put forward a GCN2 regulation mechanism, where HRSL drives the formation of activated kinase dimers. Remaining domains of GCN2 have the opposite role and in the absence of stress they help keep GCN2 basally inactive. This autoinhibitory activity is relieved upon stress ligand binding. We propose that the opposing action of HRSL and additional GCN2 domains thus yields a regulated ISR receptor. Significance statement: Regulation of protein synthesis (translation) is a central mechanism by which eukaryotic cells adapt to stressful conditions. In starving cells, this translational adaptation is achieved via the receptor kinase GCN2, which stays inactive under normal conditions, but is switched on under stress. The molecular mechanism of GCN2 switching is not well understood due to the presence of a structurally and biochemically uncharacterized h istidyl t R NA s ynthetase-like domain (HRSL) at the core of GCN2. Here we use single-particle cryo-EM and biochemistry to elucidate the structure and function of HRSL. We identify a structure at the kinase/HRSL interface, which forms crossed helices and helps position GCN2 kinase domains for activation. These data clarify the molecular mechanism of GCN2 regulation.
RESUMO
All kingdoms of life produce essential nicotinamide dinucleotide NADP(H) using NAD kinases (NADKs). A panel of published NADK structures from bacteria, eukaryotic cytosol, and yeast mitochondria revealed similar tetrameric enzymes. Here, we present the 2.8-Å structure of the human mitochondrial kinase NADK2 with a bound substrate, which is an exception to this uniformity, diverging both structurally and biochemically from NADKs. We show that NADK2 harbors a unique tetramer disruptor/dimerization element, which is conserved in mitochondrial kinases of animals (EMKA) and absent from other NADKs. EMKA stabilizes the NADK2 dimer but prevents further NADK2 oligomerization by blocking the tetramerization interface. This structural change bears functional consequences and alters the activation mechanism of the enzyme. Whereas tetrameric NADKs undergo cooperative activation via oligomerization, NADK2 is a constitutively active noncooperative dimer. Thus, our data point to a unique regulation of NADP(H) synthesis in animal mitochondria achieved via structural adaptation of the NADK2 kinase.
Assuntos
Mitocôndrias , Proteínas Mitocondriais , NAD , Fosfotransferases (Aceptor do Grupo Álcool) , Multimerização Proteica , Animais , Humanos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Mammalian cells are exquisitely sensitive to the presence of double-stranded RNA (dsRNA), a molecule that they interpret as a signal of viral presence requiring immediate attention. Upon sensing dsRNA cells activate the innate immune response, which involves transcriptional mechanisms driving inflammation and secretion of interferons (IFNs) and interferon-stimulated genes (ISGs), as well as synthesis of RNA-like signaling molecules comprised of three or more 2'-5'-linked adenylates (2-5As). 2-5As were discovered some forty years ago and described as IFN-induced inhibitors of protein synthesis. The efforts of many laboratories, aimed at elucidating the molecular mechanism and function of these mysterious RNA-like signaling oligonucleotides, revealed that 2-5A is a specific ligand for the kinase-family endonuclease RNase L. RNase L decays single-stranded RNA (ssRNA) from viruses and mRNAs (as well as other RNAs) from hosts in a process we proposed to call 2-5A-mediated decay (2-5AMD). During recent years it has become increasingly recognized that 2-5AMD is more than a blunt tool of viral RNA destruction, but a pathway deeply integrated into sensing and regulation of endogenous RNAs. Here we present an overview of recently emerged roles of 2-5AMD in host RNA regulation.
Assuntos
2',5'-Oligoadenilato Sintetase , Interações entre Hospedeiro e Microrganismos , Imunidade Inata , Estabilidade de RNA , RNA , Viroses , Vírus , Animais , Humanos , 2',5'-Oligoadenilato Sintetase/metabolismo , Regiões 3' não Traduzidas , Neoplasias da Mama , DNA Intergênico , Síndrome de Fadiga Crônica , Interferons/metabolismo , Íntrons , Retroelementos , RNA/metabolismo , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais , Viroses/imunologia , Viroses/virologia , Vírus/imunologiaRESUMO
The human microbiome encodes a large repertoire of biochemical enzymes and pathways, most of which remain uncharacterized. Here, using a metagenomics-based search strategy, we discovered that bacterial members of the human gut and oral microbiome encode enzymes that selectively phosphorylate a clinically used antidiabetic drug, acarbose1,2, resulting in its inactivation. Acarbose is an inhibitor of both human and bacterial α-glucosidases3, limiting the ability of the target organism to metabolize complex carbohydrates. Using biochemical assays, X-ray crystallography and metagenomic analyses, we show that microbiome-derived acarbose kinases are specific for acarbose, provide their harbouring organism with a protective advantage against the activity of acarbose, and are widespread in the microbiomes of western and non-western human populations. These results provide an example of widespread microbiome resistance to a non-antibiotic drug, and suggest that acarbose resistance has disseminated in the human microbiome as a defensive strategy against a potential endogenous producer of a closely related molecule.
Assuntos
Acarbose/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Inativação Metabólica , Metagenoma/genética , Boca/microbiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Acarbose/metabolismo , Amilases/metabolismo , Animais , Humanos , Hipoglicemiantes/metabolismo , Metagenoma/efeitos dos fármacos , Modelos Moleculares , Boca/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Double-stranded RNA (dsRNA), a hallmark viral material that activates antiviral interferon (IFN) responses, can appear in human cells also in the absence of viruses. We identify phosphorothioate DNAs (PS DNAs) as triggers of such endogenous dsRNA (endo-dsRNA). PS DNAs inhibit decay of nuclear RNAs and induce endo-dsRNA via accumulation of high levels of intronic and intergenic inverted retroelements (IIIR). IIIRs activate endo-dsRNA responses distinct from antiviral defense programs. IIIRs do not turn on transcriptional RIG-I/MDA5/IFN signaling, but they trigger the dsRNA-sensing pathways of OAS3/RNase L and PKR. Thus, nuclear RNA decay and nuclear-cytosolic RNA sorting actively protect from these innate immune responses to self. Our data suggest that the OAS3/RNase L and PKR arms of innate immunity diverge from antiviral IFN responses and monitor nuclear RNA decay by sensing cytosolic escape of IIIRs. OAS3 provides a receptor for IIIRs, whereas RNase L cleaves IIIR-carrying introns and intergenic RNAs.
Assuntos
Proteína DEAD-box 58/genética , Interferons/genética , Íntrons/genética , RNA de Cadeia Dupla/genética , Receptores Imunológicos/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon/genética , RNA Viral/genética , Transdução de Sinais/genéticaRESUMO
Viral and endogenous double-stranded RNA (dsRNA) is a potent trigger for programmed RNA degradation by the 2-5A/RNase L complex in cells of all mammals. This 2-5A-mediated decay (2-5AMD) is a conserved stress response switching global protein synthesis from homeostasis to production of interferons (IFNs). To understand this mechanism, we examined 2-5AMD in human cells and found that it triggers polysome collapse characteristic of inhibited translation initiation. We determined that translation initiation complexes and ribosomes purified from translation-arrested cells remain functional. However, spike-in RNA sequencing (RNA-seq) revealed cell-wide decay of basal mRNAs accompanied by rapid accumulation of mRNAs encoding innate immune proteins. Our data attribute this 2-5AMD evasion to better stability of defense mRNAs and positive feedback in the IFN response amplified by RNase L-resistant molecules. We conclude that 2-5AMD and transcription act in concert to refill mammalian cells with defense mRNAs, thereby "prioritizing" the synthesis of innate immune proteins.
Assuntos
Endorribonucleases/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica , Células A549 , Endorribonucleases/genética , Humanos , Imunidade Inata , RNA de Cadeia Dupla/genética , RNA Mensageiro/genéticaRESUMO
Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, we conducted a mass spectrometry screen and report that NOCT specifically and directly converts the dinucleotide NADP+ into NAD+ and NADPH into NADH. Further, we demonstrate that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. We obtained the 2.7 Å crystal structure of the human NOCTâ¢NADPH complex, which revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2'-terminal phosphate productively for removal. We provide evidence for NOCT targeting to mitochondria and propose that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism.
RESUMO
Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here, we developed a biosensor for 2',5'-oligoadenylate (2-5A), the natural activator of RNase L. Using this biosensor, we found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes and secreted IFNs of types I and III (IFN-ß and IFN-λ). Our data suggest that IFNs escape from the action of RNase L on translation. We propose that the 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.
Assuntos
Técnicas Biossensoriais , Endorribonucleases/química , Interferon beta/química , Interferons/química , Biossíntese de Proteínas , Linhagem Celular , Endorribonucleases/metabolismo , Humanos , Interferon beta/metabolismo , Interferons/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-AtividadeRESUMO
Nocturnin (NOCT) helps the circadian clock to adjust metabolism according to day and night activity. NOCT is upregulated in early evening and it has been proposed that NOCT serves as a deadenylase for metabolic enzyme mRNAs. We present a 2.7-Å crystal structure of the catalytic domain of human NOCT. Our structure shows that NOCT has a close overall similarity to CCR4 deadenylase family members, PDE12 and CNOT6L, and to a DNA repair enzyme TDP2. All the key catalytic residues present in PDE12, CNOT6L and TDP2 are conserved in NOCT and have the same conformations. However, we observe substantial differences in the surface properties of NOCT, an unexpectedly narrow active site pocket, and conserved structural elements in the vicinity of the catalytic center, which are unique to NOCT and absent in the deadenylases PDE12/CNOT6L. Moreover, we show that in contrast to human PDE12 and CNOT6L, NOCT is completely inactive against poly-A RNA. Our work thus reveals the structure of an intriguing circadian protein and suggests that NOCT has considerable differences from the related deadenylases, which may point to a unique cellular function of this enzyme.
Assuntos
Domínio Catalítico , Proteínas Nucleares/ultraestrutura , Fatores de Transcrição/ultraestrutura , Proteínas de Ligação a DNA , Exorribonucleases/metabolismo , Exorribonucleases/ultraestrutura , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Diester Fosfórico Hidrolases , Poli A/química , Poli A/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Ribonucleases/metabolismo , Ribonucleases/ultraestrutura , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.
Assuntos
2',5'-Oligoadenilato Sintetase/genética , Lactação/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Técnicas de Cultura de Células , Endorribonucleases/metabolismo , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Camundongos , Leite , Mutação/genética , Oligorribonucleotídeos/metabolismo , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais/genéticaRESUMO
Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of noncoding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation. Quantitative analysis of human RNA cleavage versus nascent protein synthesis in lung carcinoma cells shows that RNase L stops global translation when tRNAs, as well as rRNAs and mRNAs, are still intact. Therefore, RNase L does not have to degrade the translation machinery to stop protein synthesis. Our data point to a rapid mechanism that transforms a subtle RNA cleavage into a cell-wide translation arrest.
Assuntos
Endorribonucleases/metabolismo , Biossíntese de Proteínas , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Sítios de Ligação/genética , Linhagem Celular , Sequência Consenso , Células HeLa , Humanos , Modelos Biológicos , Clivagem do RNA , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismoRESUMO
ADAR1 isoforms are adenosine deaminases that edit and destabilize double-stranded RNA reducing its immunostimulatory activities. Mutation of ADAR1 leads to a severe neurodevelopmental and inflammatory disease of children, Aicardi-Goutiéres syndrome. In mice, Adar1 mutations are embryonic lethal but are rescued by mutation of the Mda5 or Mavs genes, which function in IFN induction. However, the specific IFN regulated proteins responsible for the pathogenic effects of ADAR1 mutation are unknown. We show that the cell-lethal phenotype of ADAR1 deletion in human lung adenocarcinoma A549 cells is rescued by CRISPR/Cas9 mutagenesis of the RNASEL gene or by expression of the RNase L antagonist, murine coronavirus NS2 accessory protein. Our result demonstrate that ablation of RNase L activity promotes survival of ADAR1 deficient cells even in the presence of MDA5 and MAVS, suggesting that the RNase L system is the primary sensor pathway for endogenous dsRNA that leads to cell death.
Assuntos
Adenosina Desaminase/deficiência , Morte Celular , Endorribonucleases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/fisiologia , Humanos , Helicase IFIH1 Induzida por Interferon/metabolismo , Proteínas de Ligação a RNARESUMO
Double-stranded RNA (dsRNA) activates the innate immune system of mammalian cells and triggers intracellular RNA decay by the pseudokinase and endoribonuclease RNase L. RNase L protects from pathogens and regulates cell growth and differentiation by destabilizing largely unknown mammalian RNA targets. We developed an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and nontargets. We show that this RNase L-dependent decay selectively affects transcripts regulated by microRNA (miR)-17/miR-29/miR-200 and other miRs that function as suppressors of mammalian cell adhesion and proliferation. RNase L mimics the effects of these miRs and acts as a suppressor of proliferation and adhesion in mammalian cells. Our data suggest that RNase L-dependent decay serves to establish an antiproliferative state via destabilization of the miR-regulated transcriptome.
Assuntos
Endorribonucleases/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Transcriptoma , Animais , Western Blotting , Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Endorribonucleases/metabolismo , Células HeLa , Humanos , Camundongos Knockout , MicroRNAs/metabolismo , Mutação , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.
Assuntos
Trifosfato de Adenosina/farmacologia , Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/antagonistas & inibidores , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Animais , Bioensaio , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Endorribonucleases/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Camundongos , Mimetismo Molecular , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição de Fator Regulador X , Radioisótopos de Enxofre , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
The mammalian innate immune system uses several sensors of double-stranded RNA (dsRNA) to develop the interferon response. Among these sensors are dsRNA-activated oligoadenylate synthetases (OAS), which produce signaling 2',5'-linked RNA molecules (2-5A) that activate regulated RNA decay in mammalian tissues. Different receptors from the OAS family contain one, two, or three copies of the 2-5A synthetase domain, which in several instances evolved into pseudoenzymes. The structures of the pseudoenzymatic domains and their roles in sensing dsRNA are unknown. Here we present the crystal structure of the first catalytically inactive domain of human OAS3 (hOAS3.DI) in complex with a 19-bp dsRNA, determined at 2.0-Å resolution. The conformation of hOAS3.DI is different from the apo- and the dsRNA-bound states of the catalytically active homolog, OAS1, reported previously. The unique conformation of hOAS3.DI disables 2-5A synthesis by placing the active site residues nonproductively, but favors the binding of dsRNA. Biochemical data show that hOAS3.DI is essential for activation of hOAS3 and serves as a dsRNA-binding module, whereas the C-terminal domain DIII carries out catalysis. The location of the dsRNA-binding domain (DI) and the catalytic domain (DIII) at the opposite protein termini makes hOAS3 selective for long dsRNA. This mechanism relies on the catalytic inactivity of domain DI, revealing a surprising role of pseudoenzyme evolution in dsRNA surveillance.
Assuntos
2',5'-Oligoadenilato Sintetase/química , RNA de Cadeia Dupla/química , Nucleotídeos de Adenina/química , Domínio Catalítico , Cristalografia por Raios X , Endorribonucleases/química , Células HeLa , Humanos , Imunidade Inata , Interferons/química , Modelos Moleculares , Oligorribonucleotídeos/química , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Estresse do Retículo Endoplasmático , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Arginina/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Análise por Conglomerados , Sequência Conservada , Citosol/metabolismo , Glicoproteínas de Membrana/química , Modelos Biológicos , Dados de Sequência Molecular , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Relação Estrutura-Atividade , Resposta a Proteínas não DobradasRESUMO
One of the hallmark mechanisms activated by type I interferons (IFNs) in human tissues involves cleavage of intracellular RNA by the kinase homology endoribonuclease RNase L. We report 2.8 and 2.1 angstrom crystal structures of human RNase L in complexes with synthetic and natural ligands and a fragment of an RNA substrate. RNase L forms a crossed homodimer stabilized by ankyrin (ANK) and kinase homology (KH) domains, which positions two kinase extension nuclease (KEN) domains for asymmetric RNA recognition. One KEN protomer recognizes an identity nucleotide (U), whereas the other protomer cleaves RNA between nucleotides +1 and +2. The coordinated action of the ANK, KH, and KEN domains thereby provides regulated, sequence-specific cleavage of viral and host RNA targets by RNase L.
Assuntos
Endorribonucleases/química , Interferon Tipo I/fisiologia , Clivagem do RNA , Estabilidade de RNA , Cristalografia por Raios X , Endorribonucleases/metabolismo , Células HeLa , Vírus da Hepatite B/genética , Humanos , Interferon Tipo I/farmacologia , Multimerização Proteica , Estrutura Terciária de Proteína , RNA Viral/químicaRESUMO
The human sensor of double-stranded RNA (dsRNA) oligoadenylate synthetase 1 (hOAS1) polymerizes ATP into 2',5'-linked iso-RNA (2-5A) involved in innate immunity, cell cycle, and differentiation. We report the crystal structure of hOAS1 in complex with dsRNA and 2'-deoxy ATP at 2.7 Å resolution, which reveals the mechanism of cytoplasmic dsRNA recognition and activation of oligoadenylate synthetases. Human OAS1 recognizes dsRNA using a previously uncharacterized protein/RNA interface that forms via a conformational change induced by binding of dsRNA. The protein/RNA interface involves two minor grooves and has no sequence-specific contacts, with the exception of a single hydrogen bond between the -NH(2) group of nucleobase G17 and the carbonyl oxygen of serine 56. Using a biochemical readout, we show that hOAS1 undergoes more than 20,000-fold activation upon dsRNA binding and that canonical or GU-wobble substitutions produce dsRNA mutants that retain either full or partial activity, in agreement with the crystal structure. Ultimately, the binding of dsRNA promotes an elaborate conformational rearrangement in the N-terminal lobe of hOAS1, which brings residues D75, D77, and D148 into proximity and creates coordination geometry for binding of two catalytic Mg(2+) ions and ATP. The assembly of this critical active-site structure provides the gate that couples binding of dsRNA to the production and downstream functions of 2-5A.
Assuntos
2',5'-Oligoadenilato Sintetase/química , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/química , 2',5'-Oligoadenilato Sintetase/metabolismo , Nucleotídeos de Adenina/química , Nucleotídeos de Adenina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Biocatálise , Cristalografia por Raios X , Citosol/metabolismo , Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxiadenina/metabolismo , Ativação Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The unfolded protein response (UPR) is a network of intracellular signaling pathways that maintain the protein-folding capacity of the endoplasmic reticulum (ER) in eukaryotic cells. Dedicated molecular sensors embedded in the ER membrane detect incompletely folded or unfolded proteins in the ER lumen and activate a transcriptional program that increases the abundance of the ER according to need. In metazoans the UPR additionally regulates translation and thus relieves unfolded protein load by globally reducing protein synthesis. If homeostasis in the ER cannot be reestablished, the metazoan UPR switches from the prosurvival to the apoptotic mode. The UPR involves a complex, coordinated action of many genes that is controlled by one ER-embedded sensor, Ire1, in yeasts, and three sensors, Ire1, PERK, and ATF6, in higher eukaryotes, including human. We discuss the emerging molecular understanding of the UPR and focus on the structural biology of Ire1 and PERK, the two recently crystallized UPR sensors.
Assuntos
Endorribonucleases/química , Proteínas de Membrana/química , Proteínas Serina-Treonina Quinases/química , Resposta a Proteínas não Dobradas , Animais , Sítios de Ligação , Endorribonucleases/fisiologia , Humanos , Proteínas de Membrana/fisiologia , Modelos Moleculares , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/fisiologia , Estrutura Quaternária de Proteína , Quercetina/química , Clivagem do RNA , Homologia Estrutural de Proteína , eIF-2 Quinase/químicaRESUMO
2',5'-linked oligoadenylates (2-5As) serve as conserved messengers of pathogen presence in the mammalian innate immune system. 2-5As induce self-association and activation of RNase L, which cleaves cytosolic RNA and promotes the production of interferons (IFNs) and cytokines driven by the transcription factors IRF-3 and NF-κB. We report that human RNase L is activated by forming high-order complexes, reminiscent of the mode of activation of the phylogenetically related transmembrane kinase/RNase Ire1 in the unfolded protein response. We describe crystal structures determined at 2.4 Å and 2.8 Å resolution, which show that two molecules of 2-5A at a time tether RNase L monomers via the ankyrin-repeat (ANK) domain. Each ANK domain harbors two distinct sites for 2-5A recognition that reside 50 Å apart. These data reveal a function for the ANK domain as a 2-5A-sensing homo-oligomerization device and describe a nonlinear, ultrasensitive regulation in the 2-5A/RNase L system poised for amplification of the IFN response.