RESUMO
Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas , Antivirais/farmacologia , Imunidade Inata , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Proteínas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , S-Adenosilmetionina , Replicação ViralRESUMO
Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.
Assuntos
Bacteroides/enzimologia , Metiltransferases/química , RNA de Transferência/química , RNA de Transferência/metabolismo , S-Adenosilmetionina/metabolismo , Compostos de Sulfidrila/metabolismo , Sulfurtransferases/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Sítios de Ligação , Biocatálise , Isopenteniladenosina/metabolismo , Metiltransferases/metabolismo , Modelos Moleculares , Domínios Proteicos , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Especificidade por Substrato , Sulfurtransferases/metabolismoRESUMO
The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 µM and are known to block the growth of Clostridium difficile1, promote hepatocellular carcinoma2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. 3). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids4; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.
Assuntos
Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/metabolismo , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiologia , Hidroxilação/genética , Redes e Vias Metabólicas/genética , Animais , Clostridium/enzimologia , Clostridium/genética , Clostridium/metabolismo , Ácido Desoxicólico/química , Ácido Desoxicólico/metabolismo , Ácido Litocólico/química , Ácido Litocólico/metabolismo , Masculino , Engenharia Metabólica , Camundongos , Óperon/genética , SimbioseRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The use of bispecific antibodies as T cell engagers can bypass the normal T cell receptor-major histocompatibility class interaction, redirect the cytotoxic activity of T cells, and lead to highly efficient tumor cell killing. However, this immunotherapy also causes significant on-target off-tumor toxicologic effects, especially when it is used to treat solid tumors. To avoid these adverse events, it is necessary to understand the fundamental mechanisms involved in the physical process of T cell engagement. We developed a multiscale computational framework to reach this goal. The framework combines simulations on the intercellular and multicellular levels. On the intercellular level, we simulated the spatial-temporal dynamics of three-body interactions among bispecific antibodies, CD3 and tumor-associated antigens (TAAs). The derived number of intercellular bonds formed between CD3 and TAAs was further transferred to the multicellular simulations as the input parameter of adhesive density between cells. Through the simulations under various molecular and cellular conditions, we were able to gain new insights into how to adopt the most appropriate strategy to maximize the drug efficacy and avoid the off-target effect. For instance, we discovered that the low antibody-binding affinity resulted in the formation of large clusters at the cell-cell interface, which could be important to control the downstream signaling pathways. We also tested different molecular architectures of the bispecific antibody and suggested the existence of an optimal length in regulating the T cell engagement. Overall, the current multiscale simulations serve as a proof-of-concept study to help in the future design of new biological therapeutics.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Humanos , Linfócitos T , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/farmacologia , Neoplasias/tratamento farmacológico , Imunoterapia/métodosRESUMO
The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed ß7-ß8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 µM). Inhibitor 6c displayed tighter binding (Kd â¼ 0.3 µM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.
RESUMO
We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.
Assuntos
COVID-19 , Humanos , Estrutura Molecular , SARS-CoV-2 , Imunidade Inata , Citosina , Redes e Vias Metabólicas , AntiviraisRESUMO
Biotin synthase (BioB) is a member of the Radical SAM superfamily of enzymes that catalyzes the terminal step of biotin (vitamin B7) biosynthesis, in which it inserts a sulfur atom in desthiobiotin to form a thiolane ring. How BioB accomplishes this difficult reaction has been the subject of much controversy, mainly around the source of the sulfur atom. However, it is now widely accepted that the sulfur atom inserted to form biotin stems from the sacrifice of the auxiliary 2Fe-2S cluster of BioB. Here, we bioinformatically explore the diversity of BioBs available in sequence databases and find an unexpected variation in the coordination of the auxiliary iron-sulfur cluster. After in vitro characterization, including the determination of biotin formation and representative crystal structures, we report a new type of BioB utilized by virtually all obligate anaerobic organisms. Instead of a 2Fe-2S cluster, this novel type of BioB utilizes an auxiliary 4Fe-5S cluster. Interestingly, this auxiliary 4Fe-5S cluster contains a ligated sulfide that we propose is used for biotin formation. We have termed this novel type of BioB, Type II BioB, with the E. coli 2Fe-2S cluster sacrificial BioB representing Type I. This surprisingly ubiquitous Type II BioB has implications for our understanding of the function and evolution of Fe-S clusters in enzyme catalysis, highlighting the difference in strategies between the anaerobic and aerobic world.
Assuntos
Proteínas de Escherichia coli , Proteínas Ferro-Enxofre , Escherichia coli/metabolismo , Biotina/química , Proteínas de Escherichia coli/química , Enxofre/química , Sulfurtransferases/metabolismo , Proteínas Ferro-Enxofre/químicaRESUMO
Change history: In the HTML version of this Letter, Extended Data Fig. 4 incorrectly corresponded to Fig. 4 (the PDF version of the figure was correct). This has been corrected online.
RESUMO
Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.
Assuntos
Antivirais/metabolismo , Citidina Trifosfato/metabolismo , Genoma Humano/genética , Proteínas/genética , Proteínas/metabolismo , Terminação da Transcrição Genética , Animais , Antivirais/química , Chlorocebus aethiops , Citidina Trifosfato/biossíntese , Citidina Trifosfato/química , Células HEK293 , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Ribonucleotídeos , Especificidade por Substrato , Células Vero , Zika virus/enzimologia , Zika virus/metabolismoRESUMO
Purine nucleoside phosphorylases (PNPs) catalyze the phosphorolysis of 6-oxypurine nucleosides with an HPO42- dianion nucleophile. Nucleosides and phosphate occupy distinct pockets in the PNP active site. Evaluation of the HPO42- site by mutagenesis, cooperative binding studies, and thermodynamic and structural analysis demonstrate that alterations in the HPO42- binding site can render PNP inactive and significantly impact subunit cooperativity and binding to transition-state analogue inhibitors. Cooperative interactions between the cationic transition-state analogue and the anionic HPO42- nucleophile demonstrate the importance of reforming the transition-state ensemble for optimal inhibition with transition-state analogues. Altered phosphate binding in the catalytic site mutants helps to explain one of the known lethal PNP deficiency syndromes in humans.
Assuntos
Purina-Núcleosídeo Fosforilase , Purinas , Humanos , Purina-Núcleosídeo Fosforilase/química , Sítios de Ligação , Domínio Catalítico , Fosfatos/químicaRESUMO
The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/virologia , Papillomaviridae/imunologia , Tomografia por Emissão de Pósitrons/métodos , Animais , Antígenos , Clonagem Molecular , Epitopos/genética , Epitopos/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/classificação , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/classificação , Imunoglobulina G/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologiaRESUMO
SUMMARY: The functional sub-string(s) of a biopolymer sequence defines the specificity of its interaction with other biomolecules and is often referred to as motifs. Computational algorithms and software have been broadly developed for finding such motifs in sequences in which the individual elements are single characters, such as those in DNA and protein sequences. However, there are more complex scenarios where the motifs exist in non-single-letter contexts, e.g. preferred patterns of chemical modifications on proteins, DNAs, RNAs or polysaccharides. To search for those motifs, we describe a new method that converts the modified sequence elements to representative single-letter codes and then uses a modified Gibbs-sampling algorithm to define the position specific scoring matrix representing the motif(s). As a proof of principle, we describe the implementation and application of an R package for discovering heparan sulfate (HS) motifs in glycan sequences, which are important in regulating protein-protein interactions. This software can be valuable for analyzing high-throughput glycoprotein binding data using microarrays with HS oligosaccharides or other biological polymers. AVAILABILITY AND IMPLEMENTATION: HSMotifDiscover is freely available as an open source R package released under an MIT license at https://github.com/bioinfoDZ/HSMotifDiscover and also available in the form of an app at https://hsmotifdiscover.shinyapps.io/HSMotifDiscover_ShinyApp/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Software , Proteínas/química , Sequência de Aminoácidos , DNA/químicaRESUMO
The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.
Assuntos
Anticorpos Antivirais/sangue , COVID-19 , SARS-CoV-2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/imunologia , COVID-19/terapia , Biologia Computacional , Diagnóstico por Computador , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto JovemRESUMO
Random amino acid copolymers used in the treatment of multiple sclerosis in man or experimental autoimmune encephalomyelitis (EAE) in mice [poly(Y,E,A,K)n, known as Copaxone, and poly(Y,F,A,K)n] function at least in part by generation of IL-10-secreting regulatory T cells that mediate bystander immunosuppression. The mechanism through which these copolymers induce Tregs is unknown. To investigate this question, four previously described Vα3.2 Vß14 T cell receptor (TCR) cDNAs, the dominant clonotype generated in splenocytes after immunization of SJL mice, that differed only in their CDR3 sequences were utilized to generate retrogenic mice. The high-level production of IL-10 as well as IL-5 and small amounts of the related cytokines IL-4 and IL-13 by CD4+ T cells isolated from the splenocytes of these mice strongly suggests that the TCR itself encodes information for specific cytokine secretion. The proliferation and production of IL-10 by these Tregs was costimulated by activation of glucocorticoid-induced TNF receptor (GITR) (expressed at high levels by these cells) through its ligand GITRL. A mechanism for generation of cells with this specificity is proposed. Moreover, retrogenic mice expressing these Tregs were protected from induction of EAE by the appropriate autoantigen.
Assuntos
Células-Tronco Hematopoéticas/citologia , Interleucina-10/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Animais , DNA Complementar , Encefalomielite Autoimune Experimental/imunologia , Feminino , Vetores Genéticos , Tolerância Imunológica , Interleucinas/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Fatores de Necrose Tumoral/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for â¼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional ß-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.
Assuntos
Leucina/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Succinatos/metabolismo , Aerossóis , Animais , Biocatálise , Ligantes , Liases/metabolismo , Malatos/metabolismo , Camundongos Endogâmicos C57BL , Filogenia , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
Queuosine (Q) is a complex tRNA modification widespread in eukaryotes and bacteria that contributes to the efficiency and accuracy of protein synthesis. Eukaryotes are not capable of Q synthesis and rely on salvage of the queuine base (q) as a Q precursor. While many bacteria are capable of Q de novo synthesis, salvage of the prokaryotic Q precursors preQ0 and preQ1 also occurs. With the exception of Escherichia coli YhhQ, shown to transport preQ0 and preQ1, the enzymes and transporters involved in Q salvage and recycling have not been well described. We discovered and characterized 2 Q salvage pathways present in many pathogenic and commensal bacteria. The first, found in the intracellular pathogen Chlamydia trachomatis, uses YhhQ and tRNA guanine transglycosylase (TGT) homologs that have changed substrate specificities to directly salvage q, mimicking the eukaryotic pathway. The second, found in bacteria from the gut flora such as Clostridioides difficile, salvages preQ1 from q through an unprecedented reaction catalyzed by a newly defined subgroup of the radical-SAM enzyme family. The source of q can be external through transport by members of the energy-coupling factor (ECF) family or internal through hydrolysis of Q by a dedicated nucleosidase. This work reinforces the concept that hosts and members of their associated microbiota compete for the salvage of Q precursors micronutrients.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/metabolismo , Clostridioides difficile/metabolismo , Infecções por Clostridium/metabolismo , Guanina/análogos & derivados , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/crescimento & desenvolvimento , Clostridioides difficile/crescimento & desenvolvimento , Infecções por Clostridium/microbiologia , Guanina/metabolismo , Humanos , Pentosiltransferases/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Transdução de Sinais , Especificidade por SubstratoRESUMO
Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.
Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Proteínas Fúngicas/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Sequência de Aminoácidos , Animais , Proteínas Arqueais/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Biocatálise , Proteínas Fúngicas/metabolismo , Humanos , Hypocrea/enzimologia , Methanomicrobiaceae/enzimologia , Camundongos , Nucleotídeos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Ligação Proteica , Especificidade por SubstratoRESUMO
3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin as part of the innate immune response. ddhCTP has been shown to act as an obligate chain terminator of flavivirus and SARS-CoV-2 RNA-dependent RNA polymerases; however, further biophysical studies have been precluded by limited access to this promising antiviral. Herein, we report a robust and scalable synthesis of ddhCTP as well as the mono- and diphosphates ddhCMP and ddhCDP, respectively. Identification of a 2'-silyl ether protection strategy enabled selective synthesis and facile purification of the 5'-triphosphate, culminating in the preparation of ddhCTP on a gram scale.
Assuntos
Antivirais , COVID-19 , Citidina Trifosfato , Humanos , Proteínas , RNA Viral , SARS-CoV-2RESUMO
Ipilimumab, a monoclonal antibody that recognizes cytotoxic T lymphocyte antigen (CTLA)-4, was the first approved "checkpoint"-blocking anticancer therapy. In mouse tumor models, the response to antibodies against CTLA-4 depends entirely on expression of the Fcγ receptor (FcγR), which may facilitate antibody-dependent cellular phagocytosis, but the contribution of simple CTLA-4 blockade remains unknown. To understand the role of CTLA-4 blockade in the complete absence of Fc-dependent functions, we developed H11, a high-affinity alpaca heavy chain-only antibody fragment (VHH) against CTLA-4. The VHH H11 lacks an Fc portion, binds monovalently to CTLA-4, and inhibits interactions between CTLA-4 and its ligand by occluding the ligand-binding motif on CTLA-4 as shown crystallographically. We used H11 to visualize CTLA-4 expression in vivo using whole-animal immuno-PET, finding that surface-accessible CTLA-4 is largely confined to the tumor microenvironment. Despite this, H11-mediated CTLA-4 blockade has minimal effects on antitumor responses. Installation of the murine IgG2a constant region on H11 dramatically enhances its antitumor response. Coadministration of the monovalent H11 VHH blocks the efficacy of a full-sized therapeutic antibody. We were thus able to demonstrate that CTLA-4-binding antibodies require an Fc domain for antitumor effect.