Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 76
Filtrar
1.
Cell ; 180(2): 278-295.e23, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31978345

RESUMO

Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adenina/metabolismo , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Cromatografia Líquida/métodos , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Espectrometria de Massas/métodos , Enzimas Multifuncionais/genética , Fosforilação , Proteínas/genética , Nucleotídeos de Purina/metabolismo , Purinas/metabolismo
3.
Nature ; 592(7853): 277-282, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33545711

RESUMO

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.


Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19/terapia , COVID-19/virologia , Evolução Molecular , Mutagênese/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Idoso , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Doença Crônica , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunização Passiva , Terapia de Imunossupressão , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Mutação , Filogenia , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de Tempo , Carga Viral/efeitos dos fármacos , Eliminação de Partículas Virais , Soroterapia para COVID-19
4.
EMBO J ; 41(24): e111179, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36341546

RESUMO

Transposable elements are a genetic reservoir from which new genes and regulatory elements can emerge. However, expression of transposable elements can be pathogenic and is therefore tightly controlled. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) recruit the co-repressor KRAB-associated protein 1 (KAP1/TRIM28) to regulate many transposable elements, but how KRAB-ZFPs and KAP1 interact remains unclear. Here, we report the crystal structure of the KAP1 tripartite motif (TRIM) in complex with the KRAB domain from a human KRAB-ZFP, ZNF93. Structure-guided mutations in the KAP1-KRAB binding interface abolished repressive activity in an epigenetic transcriptional silencing assay. Deposition of H3K9me3 over thousands of loci is lost genome-wide in cells expressing a KAP1 variant with mutations that abolish KRAB binding. Our work identifies and functionally validates the KRAB-KAP1 molecular interface, which is critical for a central transcriptional control axis in vertebrates. In addition, the structure-based prediction of KAP1 recruitment efficiency will enable optimization of KRABs used in CRISPRi.


Assuntos
Elementos de DNA Transponíveis , Proteínas Repressoras , Animais , Humanos , Proteínas Repressoras/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo , Dedos de Zinco/genética , Regulação da Expressão Gênica , Epigênese Genética
5.
EMBO Rep ; 25(3): 1310-1325, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38321165

RESUMO

Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.


Assuntos
Poxviridae , Vacínia , Humanos , Sulfatos de Condroitina , Vaccinia virus/metabolismo , Poxviridae/metabolismo , Proteínas Virais/metabolismo , Fusão de Membrana , Proteínas de Transporte
6.
Mol Cell ; 72(6): 999-1012.e6, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30449722

RESUMO

Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Long cytosolic dsRNA is recognized by MDA5. The cooperative assembly of MDA5 into helical filaments on dsRNA nucleates the assembly of a multiprotein type I interferon signaling platform. Here, we determined cryoelectron microscopy (cryo-EM) structures of MDA5-dsRNA filaments with different helical twists and bound nucleotide analogs at resolutions sufficient to build and refine atomic models. The structures identify the filament-forming interfaces, which encode the dsRNA binding cooperativity and length specificity of MDA5. The predominantly hydrophobic interface contacts confer flexibility, reflected in the variable helical twist within filaments. Mutation of filament-forming residues can result in loss or gain of signaling activity. Each MDA5 molecule spans 14 or 15 RNA base pairs, depending on the twist. Variations in twist also correlate with variations in the occupancy and type of nucleotide in the active site, providing insights on how ATP hydrolysis contributes to MDA5-dsRNA recognition.


Assuntos
Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Helicase IFIH1 Induzida por Interferon/ultraestrutura , RNA de Cadeia Dupla/ultraestrutura , Células HEK293 , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Simulação de Acoplamento Molecular , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
7.
J Biol Chem ; 300(3): 105711, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38309507

RESUMO

Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-ß responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.


Assuntos
RNA Helicases DEAD-box , Helicase IFIH1 Induzida por Interferon , RNA Helicases , RNA de Cadeia Dupla , RNA Viral , Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , Hidrólise , Imunidade Inata , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , RNA Helicases/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Humanos
8.
J Virol ; 98(2): e0177723, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38289106

RESUMO

Rubella virus encodes a nonstructural polyprotein with RNA polymerase, methyltransferase, and papain-like cysteine protease activities, along with a putative macrodomain of unknown function. Macrodomains bind ADP-ribose adducts, a post-translational modification that plays a key role in host-virus conflicts. Some macrodomains can also remove the mono-ADP-ribose adduct or degrade poly-ADP-ribose chains. Here, we report high-resolution crystal structures of the macrodomain from rubella virus nonstructural protein p150, with and without ADP-ribose binding. The overall fold is most similar to macroD-type macrodomains from various nonviral species. The specific composition and structure of the residues that coordinate ADP-ribose in the rubella virus macrodomain are most similar to those of macrodomains from alphaviruses. Isothermal calorimetry shows that the rubella virus macrodomain binds ADP-ribose in solution. Enzyme assays show that the rubella virus macrodomain can hydrolyze both mono- and poly-ADP-ribose adducts. Site-directed mutagenesis identifies Asn39 and Cys49 required for mono-ADP-ribosylhydrolase (de-MARylation) activity.IMPORTANCERubella virus remains a global health threat. Rubella infections during pregnancy can cause serious congenital pathology, for which no antiviral treatments are available. Our work demonstrates that, like alpha- and coronaviruses, rubiviruses encode a mono-ADP-ribosylhydrolase with a structurally conserved macrodomain fold to counteract MARylation by poly (ADP-ribose) polymerases (PARPs) in the host innate immune response. Our structural data will guide future efforts to develop novel antiviral therapeutics against rubella or infections with related viruses.


Assuntos
Coronavirus , Rubéola (Sarampo Alemão) , Humanos , Vírus da Rubéola/genética , Vírus da Rubéola/metabolismo , Ribose , Poli(ADP-Ribose) Polimerases/genética , Poli Adenosina Difosfato Ribose , Coronavirus/metabolismo , Adenosina Difosfato Ribose/genética , Adenosina Difosfato Ribose/metabolismo
10.
Nucleic Acids Res ; 48(18): 10313-10328, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976585

RESUMO

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR minimal core complex shows Periphilin forms an α-helical homodimer, bound by a single TASOR molecule. The NTD forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation at target loci.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas Nucleares/ultraestrutura , Proteínas de Ligação a RNA/química , Transcrição Gênica , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Epigênese Genética/genética , Inativação Gênica , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Agregados Proteicos/genética , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice , Domínios Proteicos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/ultraestrutura , Vírus/genética
11.
Proc Natl Acad Sci U S A ; 116(30): 15042-15051, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31289231

RESUMO

Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.


Assuntos
Epigênese Genética , Inativação Gênica , Proteínas Repressoras/química , Retroelementos , Proteína 28 com Motivo Tripartido/química , Sequência de Aminoácidos , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo
12.
Biochemistry ; 59(43): 4155-4162, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-32818369

RESUMO

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces TLR9 dimerization and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical framework for equilibrium ligand binding, modeling the binding of the ssDNA at the two main sites on the TLR9 ectodomain. Light scattering and fluorescence anisotropy assays performed with recombinant TLR9 ectodomain and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters, identify the likely ligand binding intermediates, and suggest cooperative modes of binding. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.


Assuntos
Receptor Toll-Like 9/química , Receptor Toll-Like 9/metabolismo , Animais , Anisotropia , Sítios de Ligação , Ilhas de CpG/fisiologia , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Difusão Dinâmica da Luz , Polarização de Fluorescência , Humanos , Hidrodinâmica , Camundongos , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo
13.
Proc Natl Acad Sci U S A ; 114(26): 6806-6811, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28607080

RESUMO

Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (ßα)6-barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first ß-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.


Assuntos
Dobramento de Proteína , Proteínas/química , Animais , Camundongos , Domínios Proteicos , Proteínas/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
14.
EMBO J ; 33(6): 542-58, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24514026

RESUMO

The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen-associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral-derived sequences efficiently induce pro-inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88-dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease.


Assuntos
Células Dendríticas/metabolismo , Imunidade Inata/imunologia , Modelos Imunológicos , Ácidos Nucleicos Heteroduplexes/metabolismo , Transdução de Sinais/imunologia , Receptor Toll-Like 9/metabolismo , Animais , Western Blotting , Células Dendríticas/imunologia , Endossomos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Polarização de Fluorescência , Imunofluorescência , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/imunologia , Ácidos Nucleicos Heteroduplexes/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Receptor Toll-Like 9/imunologia
15.
PLoS Pathog ; 12(10): e1005948, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27783673

RESUMO

Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens.


Assuntos
Virus Puumala/química , Proteínas do Envelope Viral/química , Animais , Chlorocebus aethiops , Cristalografia por Raios X , Conformação Molecular , Conformação Proteica , Células Vero
16.
Rev Med Virol ; 26(2): 115-28, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26676802

RESUMO

Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D-2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher-resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In-depth understanding of epitope composition would ensure the development of function-indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type-specific epitopes would enable rational design of cross-type neutralization via epitope re-engineering or epitope grafting in hybrid VLPs.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação de Anticorpos/imunologia , Proteínas do Capsídeo/imunologia , Papillomaviridae/imunologia , Epitopos/imunologia , Feminino , Humanos , Ligação Proteica/imunologia , Neoplasias do Colo do Útero/virologia
17.
BMC Bioinformatics ; 17: 137, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-27001666

RESUMO

BACKGROUND: Identifying key "driver" mutations which are responsible for tumorigenesis is critical in the development of new oncology drugs. Due to multiple pharmacological successes in treating cancers that are caused by such driver mutations, a large body of methods have been developed to differentiate these mutations from the benign "passenger" mutations which occur in the tumor but do not further progress the disease. Under the hypothesis that driver mutations tend to cluster in key regions of the protein, the development of algorithms that identify these clusters has become a critical area of research. RESULTS: We have developed a novel methodology, QuartPAC (Quaternary Protein Amino acid Clustering), that identifies non-random mutational clustering while utilizing the protein quaternary structure in 3D space. By integrating the spatial information in the Protein Data Bank (PDB) and the mutational data in the Catalogue of Somatic Mutations in Cancer (COSMIC), QuartPAC is able to identify clusters which are otherwise missed in a variety of proteins. The R package is available on Bioconductor at: http://bioconductor.jp/packages/3.1/bioc/html/QuartPAC.html . CONCLUSION: QuartPAC provides a unique tool to identify mutational clustering while accounting for the complete folded protein quaternary structure.


Assuntos
Proteínas/química , Interface Usuário-Computador , Algoritmos , Análise por Conglomerados , Bases de Dados de Proteínas , Humanos , Internet , Mutação , Neoplasias/genética , Neoplasias/patologia , Estrutura Quaternária de Proteína , Proteínas/genética , Proteínas/metabolismo
18.
EMBO J ; 31(7): 1714-26, 2012 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-22314235

RESUMO

Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signalling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signalling is not well understood. Here, we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16-18-basepair footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signalling model in which the CARDs on MDA5-RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry.


Assuntos
Trifosfato de Adenosina/química , RNA Helicases DEAD-box/química , RNA de Cadeia Dupla/química , Trifosfato de Adenosina/metabolismo , Animais , Cristalografia por Raios X , RNA Helicases DEAD-box/metabolismo , Helicase IFIH1 Induzida por Interferon , Camundongos , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais
19.
EMBO J ; 31(4): 919-31, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22258621

RESUMO

Toll-like receptor 9 (TLR9) recognizes microbial DNA in endolysosomal compartments. The ectodomain of TLR9 must be proteolytically cleaved by endosomal proteases to produce the active receptor capable of inducing an innate immune signal. We show that the cleaved TLR9 ectodomain is a monomer in solution and that DNA ligands with phosphodiester backbones induce TLR9 dimerization in a sequence-independent manner. Ligands with phosphorothioate (PS) backbones induce the formation of large TLR9-DNA aggregates, possibly due to the propensity of PS ligands to self-associate. DNA curvature-inducing proteins including high-mobility group box 1 and histones H2A and H2B significantly enhance TLR9 binding, suggesting that TLR9 preferentially recognizes curved DNA backbones. Our work sheds light on the molecular mechanism of TLR9 activation by endogenous protein-nucleic acid complexes, which are associated with autoimmune diseases including systemic lupus erythematosus.


Assuntos
DNA/metabolismo , Conformação de Ácido Nucleico , Receptor Toll-Like 9/metabolismo , Animais , Sequência de Bases , DNA/química , Primers do DNA , Camundongos , NF-kappa B/metabolismo , Ligação Proteica , Proteólise , Transdução de Sinais
20.
Proc Natl Acad Sci U S A ; 110(5): 1696-701, 2013 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-23319635

RESUMO

Rift Valley fever virus (RVFV), like many other Bunyaviridae family members, is an emerging human and animal pathogen. Bunyaviruses have an outer lipid envelope bearing two glycoproteins, G(N) and G(C), required for cell entry. Bunyaviruses deliver their genome into the host-cell cytoplasm by fusing their envelope with an endosomal membrane. The molecular mechanism of this key entry step is unknown. The crystal structure of RVFV G(C) reveals a class II fusion protein architecture found previously in flaviviruses and alphaviruses. The structure identifies G(C) as the effector of membrane fusion and provides a direct view of the membrane anchor that initiates fusion. A structure of nonglycosylated G(C) reveals an extended conformation that may represent a fusion intermediate. Unanticipated similarities between G(C) and flavivirus envelope proteins reveal an evolutionary link between the two virus families and provide insights into the organization of G(C) in the outer shell of RVFV.


Assuntos
Glicoproteínas de Membrana/química , Estrutura Terciária de Proteína , Vírus da Febre do Vale do Rift/metabolismo , Proteínas do Envelope Viral/química , Aminoácidos/química , Animais , Cristalografia por Raios X , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vírus da Febre do Vale do Rift/genética , Células Sf9 , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA