ABSTRACT
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.
Subject(s)
Acarbose/pharmacology , Drug Resistance, Bacterial/drug effects , Gastrointestinal Microbiome/drug effects , Hypoglycemic Agents/pharmacology , Inactivation, Metabolic , Metagenome/genetics , Mouth/microbiology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Acarbose/metabolism , Amylases/metabolism , Animals , Humans , Hypoglycemic Agents/metabolism , Metagenome/drug effects , Models, Molecular , Mouth/drug effects , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolismABSTRACT
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.
Subject(s)
Endoribonucleases/metabolism , Protein Biosynthesis , RNA Stability , RNA, Double-Stranded/metabolism , RNA, Messenger/metabolism , Transcription, Genetic , A549 Cells , Endoribonucleases/genetics , Humans , Immunity, Innate , RNA, Double-Stranded/genetics , RNA, Messenger/geneticsABSTRACT
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.
Subject(s)
Endoribonucleases/chemistry , Membrane Proteins/chemistry , Protein Serine-Threonine Kinases/chemistry , Unfolded Protein Response , Animals , Binding Sites , Endoribonucleases/physiology , Humans , Membrane Proteins/physiology , Models, Molecular , Protein Multimerization , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/physiology , Protein Structure, Quaternary , Quercetin/chemistry , RNA Cleavage , Structural Homology, Protein , eIF-2 Kinase/chemistryABSTRACT
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.
Subject(s)
Mitochondria , Mitochondrial Proteins , NAD , Phosphotransferases (Alcohol Group Acceptor) , Protein Multimerization , Animals , Humans , Mitochondria/enzymology , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , NADP/metabolism , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolismABSTRACT
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.
Subject(s)
2',5'-Oligoadenylate Synthetase , Host Microbial Interactions , Immunity, Innate , RNA Stability , RNA , Virus Diseases , Viruses , Animals , Humans , 2',5'-Oligoadenylate Synthetase/metabolism , 3' Untranslated Regions , Breast Neoplasms , DNA, Intergenic , Fatigue Syndrome, Chronic , Interferons/metabolism , Introns , Retroelements , RNA/metabolism , RNA, Double-Stranded/metabolism , Signal Transduction , Virus Diseases/immunology , Virus Diseases/virology , Viruses/immunologyABSTRACT
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.
Subject(s)
DEAD Box Protein 58/genetics , Interferons/genetics , Introns/genetics , RNA, Double-Stranded/genetics , Receptors, Immunologic/genetics , Cell Line, Tumor , HeLa Cells , Humans , Immunity, Innate/genetics , Interferon-Induced Helicase, IFIH1/genetics , RNA, Viral/genetics , Signal Transduction/geneticsABSTRACT
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.
Subject(s)
Biosensing Techniques , Endoribonucleases/chemistry , Interferon-beta/chemistry , Interferons/chemistry , Protein Biosynthesis , Cell Line , Endoribonucleases/metabolism , Humans , Interferon-beta/metabolism , Interferons/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
2',5'-Oligoadenylate Synthetase/genetics , Lactation/genetics , 2',5'-Oligoadenylate Synthetase/metabolism , Adenine Nucleotides/metabolism , Animals , Cell Culture Techniques , Endoribonucleases/metabolism , Female , Humans , Mammary Glands, Animal/metabolism , Mice , Milk , Mutation/genetics , Oligoribonucleotides/metabolism , RNA, Double-Stranded/metabolism , Signal Transduction/geneticsABSTRACT
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.
Subject(s)
Endoribonucleases/metabolism , Protein Biosynthesis , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , Binding Sites/genetics , Cell Line , Consensus Sequence , HeLa Cells , Humans , Models, Biological , RNA Cleavage , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Small Untranslated/genetics , RNA, Small Untranslated/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolismABSTRACT
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.
Subject(s)
2',5'-Oligoadenylate Synthetase/chemistry , RNA, Double-Stranded/chemistry , Adenine Nucleotides/chemistry , Catalytic Domain , Crystallography, X-Ray , Endoribonucleases/chemistry , HeLa Cells , Humans , Immunity, Innate , Interferons/chemistry , Models, Molecular , Oligoribonucleotides/chemistry , Protein Binding , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
Endoribonucleases/genetics , Gene Expression Regulation , MicroRNAs/genetics , Transcriptome , Animals , Blotting, Western , Cell Adhesion/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Cells, Cultured , Endoribonucleases/metabolism , HeLa Cells , Humans , Mice, Knockout , MicroRNAs/metabolism , Mutation , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
2',5'-Oligoadenylate Synthetase/chemistry , Nucleic Acid Conformation , Protein Structure, Tertiary , RNA, Double-Stranded/chemistry , 2',5'-Oligoadenylate Synthetase/metabolism , Adenine Nucleotides/chemistry , Adenine Nucleotides/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Biocatalysis , Crystallography, X-Ray , Cytosol/metabolism , Deoxyadenine Nucleotides/chemistry , Deoxyadenine Nucleotides/metabolism , Enzyme Activation , Humans , Models, Molecular , Molecular Sequence Data , Oligoribonucleotides/chemistry , Oligoribonucleotides/metabolism , Protein Binding , RNA, Double-Stranded/metabolism , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
Deficiencies in the protein-folding capacity of the endoplasmic reticulum (ER) in all eukaryotic cells lead to ER stress and trigger the unfolded protein response (UPR). ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the messenger RNA encoding a key transcription activator, Hac1 in yeast or XBP1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base-pairing interaction between the HAC1 intron and the HAC1 5' untranslated region. After excision of the intron, transfer RNA ligase joins the severed exons, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue. Hac1 in turn drives the UPR gene expression program comprising 7-8% of the yeast genome to counteract ER stress. Here we show that, on activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3' untranslated region. Disruption of either Ire1 clustering or HAC1 mRNA recruitment impairs UPR signalling. The HAC1 3' untranslated region element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfils this requirement for HAC1 mRNA. Recruitment of mRNA to signalling centres provides a new paradigm for the control of eukaryotic gene expression.
Subject(s)
Endoplasmic Reticulum/metabolism , RNA, Fungal/metabolism , RNA, Messenger/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Signal Transduction , Stress, Physiological , 3' Untranslated Regions/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Conserved Sequence , Gene Expression Regulation, Fungal/genetics , Introns/genetics , Membrane Glycoproteins/metabolism , Protein Biosynthesis , Protein Serine-Threonine Kinases/metabolism , RNA Splicing , RNA, Fungal/genetics , RNA, Messenger/genetics , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Stress, Physiological/geneticsABSTRACT
Aberrant folding of proteins in the endoplasmic reticulum activates the bifunctional transmembrane kinase/endoribonuclease Ire1. Ire1 excises an intron from HAC1 messenger RNA in yeasts and Xbp1 messenger RNA in metozoans encoding homologous transcription factors. This non-conventional mRNA splicing event initiates the unfolded protein response, a transcriptional program that relieves the endoplasmic reticulum stress. Here we show that oligomerization is central to Ire1 function and is an intrinsic attribute of its cytosolic domains. We obtained the 3.2-A crystal structure of the oligomer of the Ire1 cytosolic domains in complex with a kinase inhibitor that acts as a potent activator of the Ire1 RNase. The structure reveals a rod-shaped assembly that has no known precedence among kinases. This assembly positions the kinase domain for trans-autophosphorylation, orders the RNase domain, and creates an interaction surface for binding of the mRNA substrate. Activation of Ire1 through oligomerization expands the mechanistic repertoire of kinase-based signalling receptors.
Subject(s)
Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Protein Folding , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/enzymology , Basic-Leucine Zipper Transcription Factors/genetics , Binding Sites , Crystallography, X-Ray , Cytosol/metabolism , Enzyme Activation/drug effects , Introns/genetics , Membrane Glycoproteins/antagonists & inhibitors , Models, Molecular , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Denaturation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Multimerization , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Structure, Quaternary , Protein Structure, Tertiary , Repressor Proteins/genetics , Ribonucleases/chemistry , Ribonucleases/metabolism , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
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.
ABSTRACT
Accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), an intracellular signaling pathway that adjusts the protein folding capacity of the ER according to need. If homeostasis in the ER protein folding environment cannot be reestablished, cells commit to apoptosis. The ER-resident transmembrane kinase-endoribonuclease inositol-requiring enzyme 1 (IRE1) is the best characterized UPR signal transduction molecule. In yeast, Ire1 oligomerizes upon activation in response to an accumulation of misfolded proteins in the ER. Here we show that the salient mechanistic features of IRE1 activation are conserved: mammalian IRE1 oligomerizes in the ER membrane and oligomerization correlates with the onset of IRE1 phosphorylation and RNase activity. Moreover, the kinase/RNase module of human IRE1 activates cooperatively in vitro, indicating that formation of oligomers larger than four IRE1 molecules takes place upon activation. High-order IRE1 oligomerization thus emerges as a conserved mechanism of IRE1 signaling. IRE1 signaling attenuates after prolonged ER stress. IRE1 then enters a refractive state even if ER stress remains unmitigated. Attenuation includes dissolution of IRE1 clusters, IRE1 dephosphorylation, and decline in endoribonuclease activity. Thus IRE1 activity is governed by a timer that may be important in switching the UPR from the initially cytoprotective phase to the apoptotic mode.
Subject(s)
Endoplasmic Reticulum/metabolism , Endoribonucleases/metabolism , Membrane Proteins/metabolism , Protein Multimerization , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Stress, Physiological , Animals , Cells, Cultured , Endoribonucleases/chemistry , Homeostasis , Humans , Membrane Proteins/chemistry , Membrane Proteins/deficiency , Mice , Mice, Knockout , Models, Molecular , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/deficiency , Protein Structure, Quaternary , Protein Structure, TertiaryABSTRACT
BACKGROUND: The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. RESULTS: This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. CONCLUSIONS: Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.
Subject(s)
Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , RNA Cleavage/physiology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Endoribonucleases/chemistry , Endoribonucleases/genetics , Endoribonucleases/metabolism , Membrane Glycoproteins/genetics , Protein Serine-Threonine Kinases/genetics , RNA Cleavage/genetics , Saccharomyces cerevisiae Proteins/genetics , Unfolded Protein Response/genetics , Unfolded Protein Response/physiologyABSTRACT
BACKGROUND: Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity. RESULTS: Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal ß-phosphate group of a potent cofactor ADP by sulfur results in ADPßS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn2+ and Cd2+, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors. CONCLUSIONS: Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on protein-protein interactions involving Ire1's kinase domain suggests that protein kinases and pseudokinases encoded in metazoan genomes may use ATP pocket-binding ligands similarly to exert signaling roles other than phosphoryl transfer.
Subject(s)
Endoribonucleases/chemistry , Endoribonucleases/metabolism , Ribonucleases/metabolism , Crystallography, X-Ray , Endoribonucleases/isolation & purification , Protein Binding , Protein Conformation , Protein Folding , Ribonucleases/isolation & purificationABSTRACT
Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of â¼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein-RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes.
Subject(s)
Catalytic Domain , Fungal Proteins/chemistry , RNA/chemistry , Ribonucleases/chemistry , Algorithms , Amino Acid Substitution , Animals , Base Sequence , Binding Sites/genetics , Biocatalysis , Fungal Proteins/genetics , Fungal Proteins/metabolism , Kinetics , Models, Molecular , Mutation , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , RNA/genetics , RNA/metabolism , Rats , Ribonucleases/genetics , Ribonucleases/metabolism , Substrate SpecificityABSTRACT
Alpha-sarcin ribotoxins comprise a unique family of ribonucleases that cripple the ribosome by catalyzing endoribonucleolytic cleavage of ribosomal RNA at a specific location in the sarcin/ricin loop (SRL). The SRL structure alone is cleaved site-specifically by the ribotoxin, but the ribosomal context enhances the reaction rate by several orders of magnitude. We show that, for the alpha-sarcin-like ribotoxin restrictocin, this catalytic advantage arises from favorable electrostatic interactions with the ribosome. Restrictocin binds at many sites on the ribosomal surface and under certain conditions cleaves the SRL with a second-order rate constant of 1.7 x 10(10) M(-1) s(-1), a value that matches the predicted frequency of random restrictocin-ribosome encounters. The results suggest a mechanism of target location whereby restrictocin encounters ribosomes randomly and diffuses within the ribosomal electrostatic field to the SRL. These studies show a role for electrostatics in protein-ribosome recognition.