RESUMO
The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Endorribonucleases , Proteínas de Choque Térmico , Ligação Proteica , Proteínas Serina-Treonina Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Chaperona BiP do Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/química , Endorribonucleases/metabolismo , Endorribonucleases/química , Humanos , Retículo Endoplasmático/metabolismo , Resposta a Proteínas não Dobradas , Multimerização Proteica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/química , Dobramento de Proteína , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Domínios ProteicosRESUMO
Various approaches have been developed to target RNA and modulate its function with modes of action including binding and cleavage. Herein, we explored how small molecule binding is correlated with cleavage induced by heterobifunctional ribonuclease targeting chimeras (RiboTACs), where RNase L is recruited to cleave the bound RNA target, in a transcriptome-wide, unbiased fashion. Only a fraction of bound targets was cleaved by RNase L, induced by RiboTAC binding. Global analysis suggested that (i) cleaved targets generally form a region of stable structure that encompasses the small molecule binding site; (ii) cleaved targets have preferred RNase L cleavage sites nearby small molecule binding sites; (iii) RiboTACs facilitate a cellular interaction between cleaved targets and RNase L; and (iv) the expression level of the target influences the extent of cleavage observed. In one example, we converted a binder of LGALS1 (galectin-1) mRNA into a RiboTAC. In MDA-MB-231 cells, the binder had no effect on galectin-1 protein levels, while the RiboTAC cleaved LGALS1 mRNA, reduced galectin-1 protein abundance, and affected galectin-1-associated oncogenic cellular phenotypes. Using LGALS1, we further assessed additional factors including the length of the linker that tethers the two components of the RiboTAC, cellular uptake, and the RNase L-recruiting module on RiboTAC potency. Collectively, these studies may facilitate triangulation of factors to enable the design of RiboTACs.
Assuntos
Transcriptoma , Humanos , Linhagem Celular Tumoral , Endorribonucleases/metabolismo , Endorribonucleases/química , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Ribonucleases/metabolismo , Ribonucleases/químicaRESUMO
RNA helicases-central enzymes in RNA metabolism-often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa, the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-IDRRhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-IDRRhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.
Assuntos
Proteínas de Bactérias , Endorribonucleases , Proteínas Intrinsicamente Desordenadas , Pseudomonas aeruginosa , RNA Helicases , RNA Helicases/metabolismo , RNA Helicases/genética , RNA Helicases/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Endorribonucleases/metabolismo , Endorribonucleases/química , Endorribonucleases/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Ligação ProteicaRESUMO
In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurB and MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance.
Assuntos
Capuzes de RNA , Uridina Difosfato N-Acetilglicosamina , Uridina Difosfato N-Acetilglicosamina/metabolismo , Capuzes de RNA/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/química , Humanos , Escherichia coli/genética , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas ViraisRESUMO
Oligoadenylate synthetase 3 (OAS3) and ribonuclease L (RNase L) are components of a pathway that combats viral infection in mammals. Upon detection of viral double-stranded RNA (dsRNA), OAS3 synthesizes 2'-5'-oligo(A), which activates the RNase domain of RNase L by promoting the homodimerization and oligomerization of RNase L monomers. Activated RNase L rapidly degrades all cellular mRNAs, shutting off several cellular processes. We sought to understand the molecular mechanisms underlying the rapid activation of RNase L in response to viral infection. Through superresolution microscopy and live-cell imaging, we showed that OAS3 and RNase L concentrated into higher-order cytoplasmic complexes known as dsRNA-induced foci (dRIF) in response to dsRNA or infection with dengue virus, Zika virus, or West Nile virus. The concentration of OAS3 and RNase L at dRIF corresponded with the activation of RNase L-mediated RNA decay. We showed that dimerized/oligomerized RNase L concentrated in a liquid-like shell surrounding a core OAS3-dRIF structure and dynamically exchanged with the cytosol. These data establish that the condensation of dsRNA, OAS3, and RNase L into dRIF is a molecular switch that promotes the rapid activation of RNase L upon detection of dsRNA in mammalian cells.
Assuntos
2',5'-Oligoadenilato Sintetase , Endorribonucleases , RNA de Cadeia Dupla , Zika virus , Endorribonucleases/metabolismo , Endorribonucleases/genética , Endorribonucleases/química , Humanos , 2',5'-Oligoadenilato Sintetase/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/química , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , Zika virus/metabolismo , Animais , Vírus da Dengue/metabolismo , RNA Viral/metabolismo , RNA Viral/genética , Estabilidade de RNA , Vírus do Nilo Ocidental/metabolismo , Vírus do Nilo Ocidental/genética , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia , Ativação Enzimática , Células HeLa , Células HEK293RESUMO
The ability of obstacles in cellular transcripts to protect downstream but not upstream sites en masse from attack by RNase E has prompted the hypothesis that this mRNA-degrading endonuclease may scan 5'-monophosphorylated RNA linearly for cleavage sites, starting at the 5' end. However, despite its proposed regulatory importance, the migration of RNase E on RNA has never been directly observed. We have now used single-molecule FRET to monitor the dynamics of this homotetrameric enzyme on RNA. Our findings reveal that RNase E slides along unpaired regions of RNA without consuming a molecular source of energy such as ATP and that its forward progress can be impeded when it encounters a large structural discontinuity. This movement, which is bidirectional, occurs in discrete steps of variable length and requires an RNA ligand much longer than needed to occupy a single RNase E subunit. These results indicate that RNase E scans for cleavage sites by one-dimensional diffusion and suggest a possible molecular mechanism.
Assuntos
Endorribonucleases , Transferência Ressonante de Energia de Fluorescência , RNA , Endorribonucleases/metabolismo , Endorribonucleases/química , RNA/metabolismo , RNA/química , Difusão , Imagem Individual de Molécula/métodos , Trifosfato de Adenosina/metabolismo , Conformação de Ácido NucleicoRESUMO
RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world.
Assuntos
Endorribonucleases , Evolução Molecular , Subunidades Proteicas , RNA Catalítico , Ribonuclease P , Coenzimas , Endorribonucleases/química , Endorribonucleases/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ribonuclease P/química , Ribonuclease P/metabolismo , Processamento Pós-Transcricional do RNA , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Especificidade por Substrato , Eucariotos/enzimologiaRESUMO
Fungal ribotoxins are extracellular RNases that inactivate ribosomes by cleaving a single phosphodiester bond at the universally conserved sarcin-ricin loop of the large rRNA. However, to reach the ribosomes, they need to cross the plasma membrane. It is there where these toxins show their cellular specificity, being especially active against tumoral or virus-infected cells. Previous studies have shown that fungal ribotoxins interact with negatively charged membranes, typically containing phosphatidylserine or phosphatidylglycerol. This ability is rooted on their long, non-structured, positively charged loops, and its N-terminal ß-hairpin. However, its effect on complex lipid mixtures, including sphingophospholipids or cholesterol, remains poorly studied. Here, wild-type α-sarcin was used to evaluate its interaction with a variety of membranes not assayed before, which resemble much more closely mammalian cell membranes. The results confirm that α-sarcin is particularly sensitive to charge density on the vesicle surface. Its ability to induce vesicle aggregation is strongly influenced by both the lipid headgroup and the degree of saturation of the fatty acid chains. Acyl chain length is indeed particularly important for lipid mixing. Finally, cholesterol plays an important role in diluting the concentration of available negative charges and modulates the ability of α-sarcin to cross the membrane.
Assuntos
Endorribonucleases , Proteínas Fúngicas , Colesterol , Endorribonucleases/química , Proteínas Fúngicas/química , LipídeosRESUMO
Bacteria harbor diverse mechanisms to defend themselves against their viral predators, bacteriophages. In response, phages can evolve counter-defense systems, most of which are poorly understood. In T4-like phages, the gene tifA prevents bacterial defense by the type III toxin-antitoxin (TA) system toxIN, but the mechanism by which TifA inhibits ToxIN remains unclear. Here, we show that TifA directly binds both the endoribonuclease ToxN and RNA, leading to the formation of a high molecular weight ribonucleoprotein complex in which ToxN is inhibited. The RNA binding activity of TifA is necessary for its interaction with and inhibition of ToxN. Thus, we propose that TifA inhibits ToxN during phage infection by trapping ToxN on cellular RNA, particularly the abundant 16S rRNA, thereby preventing cleavage of phage transcripts. Taken together, our results reveal a novel mechanism underlying inhibition of a phage-defensive RNase toxin by a small, phage-encoded protein.
Assuntos
Bacteriófagos , Sistemas Toxina-Antitoxina , Antitoxinas/genética , Bacteriófagos/metabolismo , Endorribonucleases/genética , Endorribonucleases/química , RNA Ribossômico 16SRESUMO
Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cAn) signaling to activate a diverse range of auxiliary proteins that reinforce the CRISPR-Cas defense. Here we characterize a class of cAn-dependent effector proteins named CRISPR-Cas-associated messenger RNA (mRNA) interferase 1 (Cami1) consisting of a CRISPR-associated Rossmann fold sensor domain fused to winged helix-turn-helix and a RelE-family mRNA interferase domain. Upon activation by cyclic tetra-adenylate (cA4), Cami1 cleaves mRNA exposed at the ribosomal A-site thereby depleting mRNA and leading to cell growth arrest. The structures of apo-Cami1 and the ribosome-bound Cami1-cA4 complex delineate the conformational changes that lead to Cami1 activation and the mechanism of Cami1 binding to a bacterial ribosome, revealing unexpected parallels with eukaryotic ribosome-inactivating proteins.
Assuntos
Bactérias , Proteínas de Bactérias , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Endorribonucleases , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/classificação , RNA Mensageiro/química , Transdução de Sinais , Endorribonucleases/química , Domínios ProteicosRESUMO
Transposable elements are genomic parasites that expand within and spread between genomes1. PIWI proteins control transposon activity, notably in the germline2,3. These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m7G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors4, and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity5, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Endorribonucleases , RNA de Interação com Piwi , Animais , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Elementos de DNA Transponíveis/genética , Endorribonucleases/química , Endorribonucleases/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , RNA de Interação com Piwi/química , RNA de Interação com Piwi/genética , RNA de Interação com Piwi/metabolismo , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismoRESUMO
Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings.
Assuntos
Endorribonucleases , Eucariotos , Precursores de RNA , Splicing de RNA , RNA de Transferência , Humanos , Íntrons/genética , Conformação de Ácido Nucleico , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Especificidade por Substrato , Eucariotos/enzimologia , Endorribonucleases/química , Endorribonucleases/metabolismoRESUMO
Human ribonuclease (RNase) 1 and bovine RNase A are the proto-types of the secretory "pancreatic-type" (pt)-RNase super-family. RNase A can oligomerize through the 3D domain swapping (DS) mechanism upon acetic acid (HAc) lyophilisation, producing enzymatically active oligomeric conformers by swapping both N- and C-termini. Also some RNase 1 mutants were found to self-associate through 3D-DS, however forming only N-swapped dimers. Notably, enzymatically active dimers and larger oligomers of wt-RNase 1 were collected here, in higher amount than RNase A, from HAc lyophilisation. In particular, RNase 1 self-associates through the 3D-DS of its N-terminus and, at a higher extent, of the C-terminus. Since RNase 1 is four-residues longer than RNase A, we further analyzed its oligomerization tendency in a mutant lacking the last four residues. The C-terminus role has been investigated also in amphibian onconase (ONC®), a pt-RNase that can form only a N-swapped dimer, since its C-terminus, that is three-residues longer than RNase A, is locked by a disulfide bond. While ONC mutants designed to unlock or cut this constraint were almost unable to dimerize, the RNase 1 mutant self-associated at a higher extent than the wt, suggesting a specific role of the C-terminus in the oligomerization of different RNases. Overall, RNase 1 reaches here the highest ability, among pt-RNases, to extensively self-associate through 3D-DS, paving the way for new investigations on the structural and biological properties of its oligomers.
Assuntos
Ribonuclease Pancreático , Ribonucleases , Humanos , Animais , Bovinos , Ribonuclease Pancreático/química , Ribonucleases/química , Endorribonucleases/genética , Endorribonucleases/química , Domínios Proteicos , DimerizaçãoRESUMO
Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.
Assuntos
Endorribonucleases , MicroRNAs , RNA Mensageiro , Humanos , Genes jun/genética , Genes myc/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Endorribonucleases/química , Endorribonucleases/metabolismo , TranscriptomaRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped positive-stranded RNA virus which causes serious economic losses to pig industry worldwide. Type I IFN induces expression of interferon-stimulated genes 15 (ISG15) to inhibit virus replication. To survive in the host, PRRSV has evolved to antagonize the antiviral response of ISGylation. Previous studies have reported that nonstructural protein 2 of PRRSV inhibits the ISGylation and antiviral function of ISG15 depending on its ovarian tumor (OTU) domain/papain-like protease domain (PLP2). However, whether there are other PRRSV proteins inhibiting ISGylation of cellular proteins is less well understood. In this study, we first found that PRRSV Nsp11 decreased ISGylation of cellular proteins. Meanwhile, the expression level of ISG15 was significantly inhibited by Nsp11. Further mechanistic studies demonstrated that the transcription of ISG15 was reduced by endoribonuclease activity of Nsp11. Finally, we found that the Nsp11-induced degradation of ISG15 was partially relied on autophagy-lysosome system. Taken together, PRRSV Nsp11 antagonizes the antiviral response of ISG15 by its endoribonuclease activity to promote PRRSV replication. Our results reveal a novel mechanism that PRRSV inhibits ISGylation of cellular proteins and impairs host innate immune response.
Assuntos
Interferon Tipo I , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Doenças dos Suínos , Animais , Suínos , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Antivirais/farmacologia , Linhagem Celular , Endorribonucleases/genética , Endorribonucleases/química , Endorribonucleases/metabolismo , Imunidade Inata , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC50 value of 0.226 µM. Its inhibitory potencies on IRE1α kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α's role in ER stress.
Assuntos
Endorribonucleases , Proteínas Serina-Treonina Quinases , Endorribonucleases/química , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Inositol , Espécies Reativas de Oxigênio , Cisteína , Cinética , Simulação de Acoplamento Molecular , Ribonucleases/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Inibidores Enzimáticos/farmacologia , Estresse OxidativoRESUMO
The quick widespread of the coronavirus and speedy upsurge in the tally of cases demand the fast development of effective drugs. The uridine-directed endoribonuclease activity of nonstructural protein 15 (Nsp15) of the coronavirus is responsible for the invasion of the host immune system. Therefore, developing potential inhibitors against Nsp15 is a promising strategy. In this concern, the in silico approach can play a significant role, as it is fast and cost-effective in comparison to the trial and error approaches of experimental investigations. In this study, six turmeric derivatives (curcuminoids) were chosen for in silico analysis. The molecular interactions, pharmacokinetics, and drug-likeness of all the curcuminoids were measured. Further, the stability of Nsp15-curcuminoids complexes was appraised by employing molecular dynamics (MD) simulations and MM-PBSA approaches. All the molecules were affirmed to have strong interactions and pharmacokinetic profile. The MD simulations data stated that the Nsp15-curcuminoids complexes were stable during simulations. All the curcuminoids showed stable and high binding affinity, and these curcuminoids could be admitted as potential modulators for Nsp15 inhibition.
Assuntos
COVID-19 , Proteínas não Estruturais Virais , Humanos , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , SARS-CoV-2/metabolismo , Endorribonucleases/química , Endorribonucleases/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Antivirais/farmacologiaRESUMO
The RNA-binding protein tristetraprolin (TTP) is a potent activator of mRNA decay, specifically for transcripts bearing AU-rich elements (AREs) in their 3'-untranslated regions. TTP functions as a mediator for mRNA decay by interacting with the decay machinery and recruiting it to the target ARE-mRNA. In this study, we report a weak, but direct interaction between TTP and the human decapping enzyme DCP2, which impacts the stability of ARE transcripts. The TTP-DCP2 interaction is unusual as it involves intrinsically disordered regions (IDRs) of both binding partners. We show that the IDR of DCP2 has a propensity for oligomerization and liquid-liquid phase separation in vitro. Binding of TTP to DCP2 leads to its partitioning into phase-separated droplets formed by DCP2, suggesting that molecular crowding might facilitate the weak interaction between the two proteins and enable assembly of a decapping-competent mRNA-protein complex on TTP-bound transcripts in cells. Our studies underline the role of weak interactions in the cellular interaction network and their contribution towards cellular functionality.
Assuntos
Endorribonucleases/química , Estabilidade de RNA , Tristetraprolina/química , Regiões 3' não Traduzidas , Endorribonucleases/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMO
Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9 + million SARS-CoV-2 sequences revealed mutations across Nsp15's three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.
Assuntos
COVID-19 , Endorribonucleases , SARS-CoV-2 , Endorribonucleases Específicas de Uridilato , Proteínas não Estruturais Virais , COVID-19/virologia , Endorribonucleases/química , Endorribonucleases/genética , Humanos , Proteínas Recombinantes/química , SARS-CoV-2/enzimologia , Endorribonucleases Específicas de Uridilato/química , Endorribonucleases Específicas de Uridilato/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine-two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5' cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS.