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1.
Molecules ; 26(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072087

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Descoberta de Drogas , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/genética , Animais , Antivirais/uso terapêutico , Ácido Aurintricarboxílico/farmacologia , Ácido Aurintricarboxílico/uso terapêutico , COVID-19/virologia , Chlorocebus aethiops , Ácido Elágico/farmacologia , Ácido Elágico/uso terapêutico , Heparina/farmacologia , Heparina/uso terapêutico , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Internalização do Vírus/efeitos dos fármacos
2.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065885

RESUMO

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.


Assuntos
Agrobacterium tumefaciens/fisiologia , Proteínas de Fluorescência Verde/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Tabaco/microbiologia , Agrobacterium tumefaciens/genética , Clonagem Molecular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Plasmídeos/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transformação Bacteriana
3.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
4.
Nat Commun ; 12(1): 3214, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088904

RESUMO

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZCTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that previously reported in bacteria. Phylogenetic analyses of SepF and FtsZ from bacteria and archaea indicate that the two proteins may date back to the Last Universal Common Ancestor (LUCA), and we speculate that the archaeal mode of SepF/FtsZ interaction might reflect an ancestral feature. Our results provide insights into the mechanisms of archaeal cell division and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.


Assuntos
Proteínas Arqueais/metabolismo , Divisão Celular/fisiologia , Methanobrevibacter/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo Celular , Divisão Celular/genética , Sequência Conservada , Cristalografia por Raios X , Evolução Molecular , Methanobrevibacter/genética , Methanobrevibacter/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Filogenia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
5.
Nat Commun ; 12(1): 3384, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099674

RESUMO

Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational "anchor extension" methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.


Assuntos
Desenho de Fármacos , Inibidores de Histona Desacetilases/farmacologia , Peptídeos Cíclicos/farmacologia , Relação Estrutura-Atividade , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Ensaios Enzimáticos , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/isolamento & purificação , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/ultraestrutura , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/isolamento & purificação , Desacetilase 6 de Histona/ultraestrutura , Inibidores de Histona Desacetilases/química , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/ultraestrutura
6.
Nat Commun ; 12(1): 3396, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099711

RESUMO

Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with overlapping clinical features and the pathological hallmark of cytoplasmic deposits of misfolded proteins. The most frequent cause of familial forms of these diseases is a hexanucleotide repeat expansion in the non-coding region of the C9ORF72 gene that is translated into dipeptide repeat polymers. Here we show that proline/arginine repeat polymers derail protein folding by sequestering molecular chaperones. We demonstrate that proline/arginine repeat polymers inhibit the folding catalyst activity of PPIA, an abundant molecular chaperone and prolyl isomerase in the brain that is altered in amyotrophic lateral sclerosis. NMR spectroscopy reveals that proline/arginine repeat polymers bind to the active site of PPIA. X-ray crystallography determines the atomic structure of a proline/arginine repeat polymer in complex with the prolyl isomerase and defines the molecular basis for the specificity of disease-associated proline/arginine polymer interactions. The combined data establish a toxic mechanism that is specific for proline/arginine dipeptide repeat polymers and leads to derailed protein homeostasis in C9orf72-associated neurodegenerative diseases.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Proteína C9orf72/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/patologia , Peptidilprolil Isomerase/metabolismo , Esclerose Amiotrófica Lateral/genética , Arginina/genética , Arginina/metabolismo , Biopolímeros/metabolismo , Encéfalo/patologia , Domínio Catalítico , Cristalografia por Raios X , Expansão das Repetições de DNA , Dipeptídeos/genética , Demência Frontotemporal/genética , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptidilprolil Isomerase/isolamento & purificação , Peptidilprolil Isomerase/ultraestrutura , Prolina/genética , Prolina/metabolismo , Agregados Proteicos/genética , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Sequências Repetitivas de Aminoácidos/genética
7.
Nat Commun ; 12(1): 2987, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016967

RESUMO

The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC's ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Parede Celular/ultraestrutura , Sequência Conservada/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , Mutagênese , Filogenia , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Domínios Proteicos/genética , Multimerização Proteica , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
8.
Nat Commun ; 12(1): 2962, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016969

RESUMO

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.


Assuntos
DNA Topoisomerases Tipo II/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , Regulação Alostérica , Animais , Domínio Catalítico , Linhagem Celular , Microscopia Crioeletrônica , DNA/metabolismo , DNA/ultraestrutura , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/isolamento & purificação , DNA Topoisomerases Tipo II/metabolismo , Humanos , Mesocricetus , Modelos Moleculares , Nucleoproteínas , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/isolamento & purificação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
9.
Nat Commun ; 12(1): 2970, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016972

RESUMO

Activation of MAVS, an adaptor molecule in Rig-I-like receptor (RLR) signaling, is indispensable for antiviral immunity, yet the molecular mechanisms modulating MAVS activation are not completely understood. Ubiquitination has a central function in regulating the activity of MAVS. Here, we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS, promotes K63-linked polyubiquitination and subsequent aggregation of MAVS. USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV). Mice with a deficiency of USP18 are more susceptible to RNA virus infection. USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria. Our results indicate that USP18 functions as a post-translational modulator of MAVS-mediated antiviral signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infecções por Cardiovirus/imunologia , Infecções por Respirovirus/imunologia , Ubiquitina Tiolesterase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Animais , Infecções por Cardiovirus/virologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Vírus da Encefalomiocardite/imunologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional/imunologia , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Infecções por Respirovirus/virologia , Vírus Sendai/imunologia , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/isolamento & purificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/imunologia
10.
Nat Commun ; 12(1): 3172, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039996

RESUMO

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.


Assuntos
Proteínas do Tecido Nervoso/ultraestrutura , Neuropilina-1/ultraestrutura , Receptores de Superfície Celular/ultraestrutura , Semaforina-3A/ultraestrutura , Animais , Células COS , Chlorocebus aethiops , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-1/genética , Neuropilina-1/isolamento & purificação , Neuropilina-1/metabolismo , Ligação Proteica/genética , Domínios Proteicos/genética , Multimerização Proteica/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Semaforina-3A/genética , Semaforina-3A/isolamento & purificação , Semaforina-3A/metabolismo
11.
Nat Commun ; 12(1): 3231, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050142

RESUMO

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.


Assuntos
Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação Alostérica/genética , Animais , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genoma Fúngico , Histonas/genética , Histonas/isolamento & purificação , Humanos , Larva/genética , Larva/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
12.
Nat Commun ; 12(1): 3239, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050165

RESUMO

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.


Assuntos
Proteases Dependentes de ATP/ultraestrutura , Proteínas Mitocondriais/ultraestrutura , Proteases Dependentes de ATP/antagonistas & inibidores , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Envelhecimento/metabolismo , Bortezomib/farmacologia , Domínio Catalítico/efeitos dos fármacos , Microscopia Crioeletrônica , Ensaios Enzimáticos , Humanos , Hidrólise , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Proteólise , Proteostase , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
13.
Nat Commun ; 12(1): 3226, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050170

RESUMO

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.


Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/ultraestrutura , Ácido Fítico/metabolismo , Vírus do Sarcoma de Rous/ultraestrutura , Montagem de Vírus , Capsídeo/metabolismo , Proteínas do Capsídeo/isolamento & purificação , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Multimerização Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Vírus do Sarcoma de Rous/patogenicidade , Vírus do Sarcoma de Rous/fisiologia , Imagem Individual de Molécula , Transfecção , Liberação de Vírus
14.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947016

RESUMO

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher's) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name "B. belcheri EAS/AOS" (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.


Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Anfioxos/enzimologia , Alcadienos/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Peróxido de Hidrogênio/metabolismo , Cinética , Anfioxos/genética , Oxilipinas/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato
15.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947048

RESUMO

Hemistepta lyrata (Bunge) Bunge is a biennial medicinal plant possessing beneficial effects including anti-inflammation, and hemistepsin A (HsA) isolated from H. lyrata has been known as a hepatoprotective sesquiterpene lactone. In this report, we explored the cytotoxic effects of H. lyrata on hepatocellular carcinoma (HCC) cells and investigated the associated bioactive compounds and their relevant mechanisms. From the viability results of HCC cells treated with various H. lyrata extracts, HsA was identified as the major compound contributing to the H. lyrata-mediated cytotoxicity. HsA increased expression of cleaved PARP and cells with Sub-G1 phase, Annexin V binding, and TUNEL staining, which imply HsA induces apoptosis. In addition, HsA provoked oxidative stress by decreasing the reduced glutathione/oxidized glutathione ratio and accumulating reactive oxygen species and glutathione-protein adducts. Moreover, HsA inhibited the transactivation of signal transducer and activator of transcription 3 (STAT3) by its dephosphorylation at Y705 and glutathione conjugation. Stable expression of a constitutive active mutant of STAT3 prevented the reduction of cell viability by HsA. Finally, HsA enhanced the sensitivity of sorafenib-mediated cytotoxicity by exaggerating oxidative stress and Y705 dephosphorylation of STAT3. Therefore, HsA will be a promising candidate to induce apoptosis of HCC cells via downregulating STAT3 and sensitizing conventional chemotherapeutic agents.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lactonas/farmacologia , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/biossíntese , Fator de Transcrição STAT3/biossíntese , Sesquiterpenos/farmacologia , Ativação Transcricional/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Genes Reporter , Humanos , Proteínas de Neoplasias/genética , Estresse Oxidativo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT3/genética , Sorafenibe/farmacologia
16.
Sci Rep ; 11(1): 9825, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972631

RESUMO

In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.


Assuntos
COVID-19/imunologia , COVID-19/terapia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Cavalos/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização/métodos , Imunização Passiva/métodos , Imunoglobulina G/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
17.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947152

RESUMO

Previously, we have revealed that the miR-130 family (miR-130b, miR-301a, and miR-301b) functions as an oncomiR in bladder cancer. The pharmacological inhibition of the miR-130 family molecules by the seed-targeting strategy with an 8-mer tiny locked nucleic acid (LNA) inhibits the growth, migration, and invasion of bladder cancer cells by repressing stress fiber formation. Here, we searched for a functionally advanced target sequence with LNA for the miR-130 family with low cytotoxicity and found LNA #9 (A(L)^i^i^A(L)^T(L)^T(L)^G(L)^5(L)^A(L)^5(L)^T(L)^G) as a candidate LNA. LNA #9 inhibited cell growth in vitro and in an in vivo orthotopic bladder cancer model. Proteome-wide tyrosine phosphorylation analysis suggested that the miR-130 family upregulates a wide range of receptor tyrosine kinases (RTKs) signaling via the expression of phosphorylated Src (pSrcTyr416). SILAC-based proteome analysis and a luciferase assay identified protein tyrosine phosphatase non-receptor type 1 (PTPN1), which is implicated as a negative regulator of multiple signaling pathways downstream of RTKs as a target gene of the miR-130 family. The miR-130-targeted LNA increased and decreased PTPN1 and pSrcTyr416 expressions, respectively. PTPN1 knockdown led to increased tumor properties (cell growth, invasion, and migration) and increased pSrcTyr416 expression in bladder cancer cells, suggesting that the miR-130 family upregulates multiple RTK signaling by targeting PTPN1 and subsequent Src activation in bladder cancer. Thus, our newly designed miR-130 family targeting LNA could be a promising nucleic acid therapeutic agent for bladder cancer.


Assuntos
Antineoplásicos/uso terapêutico , MicroRNAs/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Oligonucleotídeos/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , RNA Neoplásico/antagonistas & inibidores , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Camundongos , MicroRNAs/genética , RNA Neoplásico/genética , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes/metabolismo , Regulação para Cima , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Commun ; 12(1): 2843, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990585

RESUMO

Although the accessory proteins are considered non-essential for coronavirus replication, accumulating evidences demonstrate they are critical to virus-host interaction and pathogenesis. Orf9b is a unique accessory protein of SARS-CoV-2 and SARS-CoV. It is implicated in immune evasion by targeting mitochondria, where it associates with the versatile adapter TOM70. Here, we determined the crystal structure of SARS-CoV-2 orf9b in complex with the cytosolic segment of human TOM70 to 2.2 Å. A central portion of orf9b occupies the deep pocket in the TOM70 C-terminal domain (CTD) and adopts a helical conformation strikingly different from the ß-sheet-rich structure of the orf9b homodimer. Interactions between orf9b and TOM70 CTD are primarily hydrophobic and distinct from the electrostatic interaction between the heat shock protein 90 (Hsp90) EEVD motif and the TOM70 N-terminal domain (NTD). Using isothermal titration calorimetry (ITC), we demonstrated that the orf9b dimer does not bind TOM70, but a synthetic peptide harboring a segment of orf9b (denoted C-peptide) binds TOM70 with nanomolar KD. While the interaction between C-peptide and TOM70 CTD is an endothermic process, the interaction between Hsp90 EEVD and TOM70 NTD is exothermic, which underscores the distinct binding mechanisms at NTD and CTD pockets. Strikingly, the binding affinity of Hsp90 EEVD motif to TOM70 NTD is reduced by ~29-fold when orf9b occupies the pocket of TOM70 CTD, supporting the hypothesis that orf9b allosterically inhibits the Hsp90/TOM70 interaction. Our findings shed light on the mechanism underlying SARS-CoV-2 orf9b mediated suppression of interferon responses.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas de Transporte da Membrana Mitocondrial/química , Complexos Multiproteicos/química , Proteínas Recombinantes/química , Sítios de Ligação/genética , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Interações entre Hospedeiro e Microrganismos , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia
19.
Nat Commun ; 12(1): 2942, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011988

RESUMO

The association between reduced myofilament force-generating capacity (Fmax) and heart failure (HF) is clear, however the underlying molecular mechanisms are poorly understood. Here, we show impaired Fmax arises from reduced BAG3-mediated sarcomere turnover. Myofilament BAG3 expression decreases in human HF and positively correlates with Fmax. We confirm this relationship using BAG3 haploinsufficient mice, which display reduced Fmax and increased myofilament ubiquitination, suggesting impaired protein turnover. We show cardiac BAG3 operates via chaperone-assisted selective autophagy (CASA), conserved from skeletal muscle, and confirm sarcomeric CASA complex localization is BAG3/proteotoxic stress-dependent. Using mass spectrometry, we characterize the myofilament CASA interactome in the human heart and identify eight clients of BAG3-mediated turnover. To determine if increasing BAG3 expression in HF can restore sarcomere proteostasis/Fmax, HF mice were treated with rAAV9-BAG3. Gene therapy fully rescued Fmax and CASA protein turnover after four weeks. Our findings indicate BAG3-mediated sarcomere turnover is fundamental for myofilament functional maintenance.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Insuficiência Cardíaca/fisiopatologia , Miócitos Cardíacos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Modelos Animais de Doenças , Feminino , Terapia Genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcômeros/metabolismo
20.
mSphere ; 6(3)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980675

RESUMO

Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43.IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.


Assuntos
Coronavirus Humano OC43/fisiologia , Efeito Citopatogênico Viral , Receptores Virais/metabolismo , Serina Endopeptidases/metabolismo , Carga Viral/métodos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Coronavirus Humano OC43/isolamento & purificação , Humanos , Técnicas Imunoenzimáticas , Receptores Virais/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética , Células Vero/virologia , Cultura de Vírus , Internalização do Vírus , Replicação Viral
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