RESUMO
Picornaviruses are small RNA viruses that hijack host cell machinery to promote their replication. During infection, these viruses express two proteases, 2Apro and 3Cpro, which process viral proteins. They also subvert a number of host functions, including innate immune responses, host protein synthesis, and intracellular transport, by utilizing poorly understood mechanisms for rapidly and specifically targeting critical host proteins. Here, we used proteomic tools to characterize 2Apro interacting partners, functions, and targeting mechanisms. Our data indicate that, initially, 2Apro primarily targets just two cellular proteins: eukaryotic translation initiation factor eIF4G (a critical component of the protein synthesis machinery) and Nup98 (an essential component of the nuclear pore complex, responsible for nucleocytoplasmic transport). The protease appears to employ two different cleavage mechanisms; it likely interacts with eIF3L, utilizing the eIF3 complex to proteolytically access the eIF4G protein but also directly binds and degrades Nup98. This Nup98 cleavage results in only a marginal effect on nuclear import of proteins, while nuclear export of proteins and mRNAs were more strongly affected. Collectively, our data indicate that 2Apro selectively inhibits protein translation, key nuclear export pathways, and cellular mRNA localization early in infection to benefit viral replication at the expense of particular cell functions.
Assuntos
Peptídeo Hidrolases , Picornaviridae , Fator de Iniciação Eucariótico 4G/metabolismo , Peptídeo Hidrolases/metabolismo , Picornaviridae/enzimologia , Picornaviridae/genética , Proteômica , RNA Mensageiro/metabolismoRESUMO
We have undertaken a systematic structural study of Thermus thermophilus Argonaute (TtAgo) ternary complexes containing single-base bulges positioned either within the seed segment of the guide or target strands and at the cleavage site. Our studies establish that single-base bulges 7T8, 5A6 and 4A5 on the guide strand are stacked-into the duplex, with conformational changes localized to the bulge site, thereby having minimal impact on the cleavage site. By contrast, single-base bulges 6'U7' and 6'A7' on the target strand are looped-out of the duplex, with the resulting conformational transitions shifting the cleavable phosphate by one step. We observe a stable alignment for the looped-out 6'N7' bulge base, which stacks on the unpaired first base of the guide strand, with the looped-out alignment facilitated by weakened Watson-Crick and reversed non-canonical flanking pairs. These structural studies are complemented by cleavage assays that independently monitor the impact of bulges on TtAgo-mediated cleavage reaction.
Assuntos
Proteínas Argonautas/química , Proteínas de Bactérias/química , DNA Bacteriano/química , Oligodesoxirribonucleotídeos/química , Oligorribonucleotídeos/química , Thermus thermophilus/enzimologia , Motivos de Aminoácidos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Clivagem do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Oligorribonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Thermus thermophilus/genéticaRESUMO
Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2',5')pA(3',5')p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTING(H232) adopts a "closed" conformation upon binding c[G(2',5')pA(3',5')p] and its linkage isomer c[G(2',5')pA(2',5')p], as does mouse mSting(R231) on binding c[G(2',5')pA(3',5')p], c[G(3',5')pA(3',5')p] and the antiviral agent DMXAA, leading to similar "closed" conformations. Comparing hSTING to mSting, 2',5'-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3',5'-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING.
Assuntos
Antivirais/farmacologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/metabolismo , Xantonas/farmacologia , Animais , Cristalografia por Raios X , GMP Cíclico/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Mutagênese , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
We have solved the crystal structure of human ARGONAUTE1 (hAGO1) bound to endogenous 5'-phosphorylated guide RNAs. To identify changes that evolutionarily rendered hAGO1 inactive, we compared our structure with guide-RNA-containing and cleavage-active hAGO2. Aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hAGO1 introduce a kink in the cS7 loop, forming a convex surface within the hAGO1 nucleic-acid-binding channel near the inactive catalytic site. We predicted that even upon restoration of the catalytic tetrad, hAGO1-cS7 sterically hinders the placement of a fully paired guide-target RNA duplex into the endonuclease active site. Consistent with this hypothesis, reconstitution of the catalytic tetrad with R805H led to low-level hAGO1 cleavage activity, whereas combining R805H with cS7 substitutions P670S and P675Q substantially augmented hAGO1 activity. Evolutionary amino acid changes to hAGO1 were readily reversible, suggesting that loading of guide RNA and pairing of seed-based miRNA and target RNA constrain its sequence drift.
Assuntos
Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Células Eucarióticas/fisiologia , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Sequência de Aminoácidos , Proteínas Argonautas/química , Eucariotos , Células Eucarióticas/metabolismo , Fatores de Iniciação em Eucariotos/química , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos MolecularesRESUMO
Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2',5')pA and A(3',5')pG phosphodiester linkages, designated c[G(2',5')pA(3',5')p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2',5')pA(3',5')p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5'-pppG(2',5')pA prior to cyclization to c[G(2',5')pA(3',5')p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2',5')pA(3',5')p] as a founding member of a family of metazoan 2',5'-containing cyclic heterodinucleotide second messengers distinct from bacterial 3',5' cyclic dinucleotides.
Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/química , Sistemas do Segundo Mensageiro , 2',5'-Oligoadenilato Sintetase/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Camundongos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Nucleotidiltransferases/metabolismo , Alinhamento de SequênciaRESUMO
We recently reported that Drosophila Insensitive (Insv) promotes sensory organ development and has activity as a nuclear corepressor for the Notch transcription factor Suppressor of Hairless [Su(H)]. Insv lacks domains of known biochemical function but contains a single BEN domain (i.e., a "BEN-solo" protein). Our chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) analysis confirmed binding of Insensitive to Su(H) target genes in the Enhancer of split gene complex [E(spl)-C]; however, de novo motif analysis revealed a novel site strongly enriched in Insv peaks (TCYAATHRGAA). We validate binding of endogenous Insv to genomic regions bearing such sites, whose associated genes are enriched for neural functions and are functionally repressed by Insv. Unexpectedly, we found that the Insv BEN domain binds specifically to this sequence motif and that Insv directly regulates transcription via this motif. We determined the crystal structure of the BEN-DNA target complex, revealing homodimeric binding of the BEN domain and extensive nucleotide contacts via α helices and a C-terminal loop. Point mutations in key DNA-contacting residues severely impair DNA binding in vitro and capacity for transcriptional regulation in vivo. We further demonstrate DNA-binding and repression activities by the mammalian neural BEN-solo protein BEND5. Altogether, we define novel DNA-binding activity in a conserved family of transcriptional repressors, opening a molecular window on this extensive gene family.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/genética , Modelos Moleculares , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Correpressoras/química , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Sistema Nervoso/embriologia , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de SequênciaRESUMO
Riboswitches are mRNA regions that regulate the expression of genes in response to various cellular metabolites. These RNA sequences, typically situated in the untranslated regions of mRNAs, possess complex structures that dictate highly specific binding to certain ligands, such as nucleobases, coenzymes, amino acids, and sugars, without protein assistance. Depending on the presence of the ligand, metabolite-binding domains of riboswitches can adopt two alternative conformations, which define the conformations of the adjacent sequences involved in the regulation of gene expression. In order to understand in detail the nature of riboswitch-ligand interactions and the molecular basis of riboswitch-based gene expression control, it is necessary to determine the three-dimensional structures of riboswitch-ligand complexes. This chapter outlines the techniques that are employed to prepare riboswitch-ligand complexes for structure determination using X-ray crystallography. The chapter describes the principles of construct design, in vitro transcription, RNA purification, complex formation, and crystallization screening utilized during the successful crystallization of several riboswitches.