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1.
Nat Commun ; 12(1): 4176, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234134

RESUMO

Mammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor µ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase µ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.


Assuntos
Proteínas do Capsídeo/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Orthoreovirus/ultraestrutura , RNA Viral/metabolismo , Fatores de Transcrição/metabolismo , Regulação Alostérica , Animais , Proteínas do Capsídeo/ultraestrutura , Linhagem Celular , Microscopia Crioeletrônica , Regulação Viral da Expressão Gênica , Genoma Viral , Macaca mulatta , Nucleosídeo-Trifosfatase/ultraestrutura , Orthoreovirus/genética , Orthoreovirus/metabolismo , Multimerização Proteica , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/ultraestrutura , RNA Mensageiro/metabolismo , RNA Viral/ultraestrutura , RNA Polimerase Dependente de RNA/metabolismo , Fatores de Transcrição/ultraestrutura , Ativação Transcricional , Montagem de Vírus/genética
2.
FEBS Lett ; 595(14): 1876-1885, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34060653

RESUMO

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Synechocystis/enzimologia , Tilacoides/enzimologia , Trifosfato de Adenosina/química , Proteínas de Bactérias/genética , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólise , Cinética , Proteínas de Membrana/genética , Microscopia Eletrônica , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Synechocystis/genética , Synechocystis/ultraestrutura , Tilacoides/genética , Tilacoides/ultraestrutura
3.
Nucleic Acids Res ; 49(1): 504-518, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33300032

RESUMO

Mitomycin repair factor A represents a family of DNA helicases that harbor a domain of unknown function (DUF1998) and support repair of mitomycin C-induced DNA damage by presently unknown molecular mechanisms. We determined crystal structures of Bacillus subtilis Mitomycin repair factor A alone and in complex with an ATP analog and/or DNA and conducted structure-informed functional analyses. Our results reveal a unique set of auxiliary domains appended to a dual-RecA domain core. Upon DNA binding, a Zn2+-binding domain, encompassing the domain of unknown function, acts like a drum that rolls out a canopy of helicase-associated domains, entrapping the substrate and tautening an inter-domain linker across the loading strand. Quantification of DNA binding, stimulated ATPase and helicase activities in the wild type and mutant enzyme variants in conjunction with the mode of coordination of the ATP analog suggest that Mitomycin repair factor A employs similar ATPase-driven conformational changes to translocate on DNA, with the linker ratcheting through the nucleotides like a 'skipping rope'. The electrostatic surface topology outlines a likely path for the displaced DNA strand. Our results reveal unique molecular mechanisms in a widespread family of DNA repair helicases linked to bacterial antibiotics resistance.


Assuntos
DNA Helicases/metabolismo , Reparo do DNA , Modelos Químicos , Nucleosídeo-Trifosfatase/metabolismo , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA/metabolismo , Dano ao DNA , DNA Helicases/química , DNA Helicases/classificação , Resistência Microbiana a Medicamentos , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Família Multigênica , Nucleosídeo-Trifosfatase/classificação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Eletricidade Estática , Relação Estrutura-Atividade , Zinco/metabolismo
4.
RNA ; 27(2): 221-233, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33219089

RESUMO

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control, remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 Å. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved methyltransferase Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality control during ribosome assembly.


Assuntos
Adenilato Quinase/genética , Regulação Fúngica da Expressão Gênica , Metiltransferases/genética , Proteínas Nucleares/genética , Nucleosídeo-Trifosfatase/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenilato Quinase/química , Adenilato Quinase/metabolismo , Sítios de Ligação , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Biogênese de Organelas , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Nat Commun ; 11(1): 6418, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33339827

RESUMO

Cellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the ß and ß' subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Subunidades Proteicas/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/química , Modelos Moleculares , Nucleosídeo-Trifosfatase/química , Multimerização Proteica , Subunidades Proteicas/química
6.
Arch Biochem Biophys ; 695: 108631, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33080173

RESUMO

Among Flaviviridae, in West Nile virus (WNV) and Hepatitis C virus (HCV), the non-structural protein NS4A modulates the NTPase activity of viral helicases during nucleic acid unwinding through its N-terminal disordered residues (1-50). In HCV, the acidic NS4A also serves as a cofactor for regulating the NS3 protease activity. However, in case of Zika virus (ZIKV), the role of NS4A and its impact on activities of NS3 helicase and protease is not known. In order to elucidate the role of NS4A, we checked the NTPase activity of NS3 helicase and protease activity of NS3 protease in presence of NS4A N-terminal region (residues 1-48) peptide. Our enzyme kinetics results together with binding experiment clearly demonstrate that NS3 helicase in presence of NS4A peptide increased the rate of ATP hydrolysis whereas the protease activity of NS3 protease was not affected. Therefore, like WNV and HCV, our results establish a role of ZIKV NS4A being a cofactor for modulating the NTPase activity of ZIKV NS3 helicase.


Assuntos
Nucleosídeo-Trifosfatase/química , RNA Helicases/química , Serina Endopeptidases/química , Proteínas Virais/química , Zika virus/enzimologia , Coenzimas , Nucleosídeo-Trifosfatase/genética , Domínios Proteicos , RNA Helicases/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Zika virus/genética
7.
J Mol Biol ; 432(20): 5544-5564, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32750390

RESUMO

A hallmark of the catalytically essential Walker B motif of P-loop NTPases is the presence of an acidic residue (aspartate/glutamate) for efficient Mg2+ coordination. Although the Walker B motif has been identified in well-studied examples of P-loop NTPases, its identity is ambiguous in many families, for example, in the prokaryotic small Ras-like GTPase family of MglA. MglA, belonging to TRAFAC class of P-loop NTPases, possesses a threonine at the position equivalent to Walker B aspartate in eukaryotic Ras-like GTPases. To resolve the identity of the Walker B residue in MglA, we carried out a comprehensive analysis of Mg2+ coordination on P-loop NTPase structures. Atoms in the octahedral coordination of Mg2+ and their interactions comprise a network including water molecules, Walker A, Walker B and switch motifs of P-loop NTPases. Based on the conserved geometry of Mg2+ coordination, we confirm that a conserved aspartate functions as the Walker B residue of MglA, and validate it through mutagenesis and biochemical characterization. Location of the newly identified aspartate is spatially equivalent to the Walker B residue of the ASCE division of P-loop NTPases. Furthermore, similar to the allosteric regulation of the Walker B aspartate conformation in MglA, we identify protein families in which large conformational changes involving Walker B motif potentially function as allosteric regulators. The study unravels conserved features of Mg2+ coordination among divergent families of P-loop NTPases, especially between ancient Ras-like GTPases and ASCE family of ATPases. The conserved geometric features provide a foundation for design of nucleotide-hydrolyzing enzymes.


Assuntos
Domínio AAA/fisiologia , Proteínas AAA/metabolismo , GTP Fosfo-Hidrolases/química , Células Procarióticas/metabolismo , Proteínas ras/química , Proteínas AAA/genética , Evolução Molecular , GTP Fosfo-Hidrolases/genética , Modelos Moleculares , Nucleosídeo-Trifosfatase/metabolismo , Conformação Proteica , Proteínas ras/genética
8.
Proc Natl Acad Sci U S A ; 117(36): 22237-22248, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839316

RESUMO

NOD-like receptors (NLRs) are traditionally recognized as major inflammasome components. The role of NLRs in germ cell differentiation and reproduction is not known. Here, we identified the gonad-specific Nlrp14 as a pivotal regulator in primordial germ cell-like cell (PGCLC) differentiation in vitro. Physiologically, knock out of Nlrp14 resulted in reproductive failure in both female and male mice. In adult male mice, Nlrp14 knockout (KO) inhibited differentiation of spermatogonial stem cells (SSCs) and meiosis, resulting in trapped SSCs in early stages, severe oligozoospermia, and sperm abnormality. Mechanistically, NLRP14 promoted spermatogenesis by recruiting a chaperone cofactor, BAG2, to bind with HSPA2 and form the NLRP14-HSPA2-BAG2 complex, which strongly inhibited ChIP-mediated HSPA2 polyubiquitination and promoted its nuclear translocation. Finally, loss of HSPA2 protection and BAG2 recruitment by NLRP14 was confirmed in a human nonsense germline variant associated with male sterility. Together, our data highlight a unique proteasome-mediated, noncanonical function of NLRP14 in PGCLC differentiation and spermatogenesis, providing mechanistic insights of gonad-specific NLRs in mammalian germline development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Espermatogênese/genética , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Células-Tronco Germinativas Adultas/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Feminino , Deleção de Genes , Regulação da Expressão Gênica/fisiologia , Variação Genética , Células Germinativas , Proteínas de Choque Térmico HSP70/genética , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Chaperonas Moleculares/genética , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Espermatogênese/fisiologia
9.
Virol Sin ; 35(3): 321-329, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32500504

RESUMO

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.


Assuntos
Betacoronavirus/metabolismo , Bismuto/farmacologia , Metiltransferases/metabolismo , Nucleosídeo-Trifosfatase/efeitos dos fármacos , RNA Helicases/efeitos dos fármacos , Sais/farmacologia , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Betacoronavirus/enzimologia , Betacoronavirus/genética , COVID-19 , Infecções por Coronavirus/virologia , Humanos , Metiltransferases/genética , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Pandemias , Pneumonia Viral/virologia , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes , SARS-CoV-2 , Síndrome Respiratória Aguda Grave , Proteínas não Estruturais Virais/genética , Replicação Viral
10.
Nat Commun ; 11(1): 3165, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576829

RESUMO

SAMHD1 regulates cellular 2'-deoxynucleoside-5'-triphosphate (dNTP) homeostasis by catalysing the hydrolysis of dNTPs into 2'-deoxynucleosides and triphosphate. In CD4+ myeloid lineage and resting T-cells, SAMHD1 blocks HIV-1 and other viral infections by depletion of the dNTP pool to a level that cannot support replication. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome and hypermutated cancers. Furthermore, SAMHD1 sensitises cancer cells to nucleoside-analogue anti-cancer therapies and is linked with DNA repair and suppression of the interferon response to cytosolic nucleic acids. Nevertheless, despite its requirement in these processes, the fundamental mechanism of SAMHD1-catalysed dNTP hydrolysis remained unknown. Here, we present structural and enzymological data showing that SAMHD1 utilises an active site, bi-metallic iron-magnesium centre that positions a hydroxide nucleophile in-line with the Pα-O5' bond to catalyse phosphoester bond hydrolysis. This precise molecular mechanism for SAMHD1 catalysis, reveals how SAMHD1 down-regulates cellular dNTP and modulates the efficacy of nucleoside-based anti-cancer and anti-viral therapies.


Assuntos
Nucleosídeo-Trifosfatase/química , Proteína 1 com Domínio SAM e Domínio HD/química , Água/química , Doenças Autoimunes do Sistema Nervoso/metabolismo , Domínio Catalítico , Cristalografia por Raios X , HIV-1/genética , HIV-1/fisiologia , Humanos , Hidrólise , Interferons , Modelos Moleculares , Mutação , Malformações do Sistema Nervoso/metabolismo , Polifosfatos , Conformação Proteica , Proteína 1 com Domínio SAM e Domínio HD/genética , Replicação Viral/fisiologia
11.
Parasitol Res ; 119(8): 2587-2595, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32524267

RESUMO

Lycorine is an Amaryllidaceae alkaloid that presents anti-Trichomonas vaginalis activity. T. vaginalis causes trichomoniasis, the most common non-viral sexually transmitted infection. The modulation of T. vaginalis purinergic signaling through the ectonucleotidases, nucleoside triphosphate diphosphohydrolase (NTPDase), and ecto-5'-nucleotidase represents new targets for combating the parasite. With this knowledge, the aim of this study was to investigate whether NTPDase and ecto-5'-nucleotidase inhibition by lycorine could lead to extracellular ATP accumulation. Moreover, the lycorine effect on the reactive oxygen species (ROS) production by neutrophils and parasites was evaluated as well as the alkaloid toxicity. The metabolism of purines was assessed by HPLC. ROS production was measured by flow cytometry. Cytotoxicity against epithelial vaginal cells and fibroblasts was tested, as well as the hemolytic effect of lycorine and its in vivo toxicity in Galleria mellonella larvae. Our findings showed that lycorine caused ATP accumulation due to NTPDase inhibition. The alkaloid did not affect the ROS production by T. vaginalis; however, it increased ROS levels in neutrophils incubated with lycorine-treated trophozoites. Lycorine was cytotoxic against vaginal epithelial cells and fibroblasts; conversely, it was not hemolytic neither exhibited toxicity against the in vivo model of G. mellonella larvae. Overall, besides having anti-T. vaginalis activity, lycorine modulates ectonucleotidases and stimulates neutrophils to secrete ROS. This mechanism of action exerted by the alkaloid could enhance the susceptibility of T. vaginalis to host immune cell, contributing to protozoan clearance.


Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Amaryllidaceae/química , Antiprotozoários/farmacologia , Neutrófilos/metabolismo , Nucleosídeo-Trifosfatase/antagonistas & inibidores , Fenantridinas/farmacologia , Extratos Vegetais/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Tricomoníase/metabolismo , Trichomonas vaginalis/enzimologia , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/metabolismo , Humanos , Neutrófilos/efeitos dos fármacos , Nucleosídeo-Trifosfatase/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tricomoníase/parasitologia , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/crescimento & desenvolvimento , Trichomonas vaginalis/metabolismo , Trofozoítos/efeitos dos fármacos , Trofozoítos/enzimologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismo
12.
Viruses ; 12(6)2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32585808

RESUMO

African swine fever virus (ASFV) is the causative agent of the African swine fever (ASF) epizootic currently affecting pigs throughout Eurasia, causing significant economic losses in the swine industry. The virus genome encodes for more than 160 genes, of which only a few have been studied in detail. Here we describe the previously uncharacterized ASFV open reading frame (ORF) C962R, a gene encoding for a putative NTPase. RNA transcription studies using infected swine macrophages demonstrate that the C962R gene is translated as a late virus protein. A recombinant ASFV lacking the C962R gene (ASFV-G-ΔC962R) demonstrates in vivo that the C962R gene is non-essential, since ASFV-G-ΔC962R has similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔC962R produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the C962R gene from the ASFV genome does not impact virulence.


Assuntos
Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/patogenicidade , Febre Suína Africana/patologia , Nucleosídeo-Trifosfatase/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Deleção de Genes , Genoma Viral/genética , Macrófagos/virologia , Fases de Leitura Aberta/genética , Alinhamento de Sequência , Suínos , Doenças dos Suínos/virologia , Proteínas Virais/genética , Fatores de Virulência/genética , Replicação Viral/genética
13.
Mar Drugs ; 18(3)2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32183121

RESUMO

Toxoplasma gondii is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. Neospora caninum, another type of protozoa, is closely related to Toxoplasma gondii. Infections of the protozoa in animals also causes serious diseases such as Encephalomyelitis and Myositis-Polyradiculitis in dogs or abortion in cows. Both Toxoplasma gondii and Neospora caninum have similar nucleoside triphosphate hydrolases (NTPase), NcNTPase and TgNTPase-I in Neospora caninum and Toxoplasma gondii, respectively. These possibly play important roles in propagation and survival. Thus, we targeted the enzymes for drug discovery and tried to establish a novel high-standard assay by a combination of original biochemical enzyme assay and fluorescent assay to determine ADP content. We then validated whether or not it can be applied to high-throughput screening (HTS). Then, it fulfilled criterion to carry out HTS in both of the enzymes. In order to identify small molecules having inhibitory effects on the protozoan enzyme, we also performed HTS using two synthetic compound libraries and an extract library derived from marine bacteria and then, identified 19 compounds and 6 extracts. Nagasaki University collected many extracts from over 18,000 marine bacteria found in local Omura bay, and continues to compile an extensive collection of synthetic compounds from numerous drug libraries established by Japanese chemists.


Assuntos
Medições Luminescentes , Neospora/enzimologia , Nucleosídeo-Trifosfatase/análise , Toxoplasma/enzimologia , Animais , Humanos
14.
Arch Biochem Biophys ; 685: 108350, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32220566

RESUMO

Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/química , Hidrólise , Mutagênese Sítio-Dirigida , Mutação , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Potássio/metabolismo , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato
15.
Structure ; 28(6): 604-612.e3, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32049031

RESUMO

Members of the family Reoviridae package several copies of the viral polymerase complex into their capsid to carry out replication and transcription within viral particles. Classical single-particle reconstruction encounters difficulties resolving structures such as the intraparticle polymerase complex because refinement can converge to an incorrect map and because the map could depict a nonrepresentative subset of particles or an average of heterogeneous particles. Using the nine-segmented Fako virus, we tested hypotheses for the arrangement and number of polymerase complexes within the virion by measuring how well each hypothesis describes the set of cryoelectron microscopy images of individual viral particles. We find that the polymerase complex in Fako virus binds at ten possible sites despite having only nine genome segments. A single asymmetric configuration describes the arrangement of these complexes in both virions and genome-free capsids. Similarities between the arrangements of Reoviridae with 9, 10, and 11 segments indicate the generalizability of this architecture.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Reoviridae/ultraestrutura , Animais , Linhagem Celular , Microscopia Crioeletrônica , Modelos Moleculares , Conformação Proteica , Reoviridae/química , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
16.
J Biomol Struct Dyn ; 38(16): 4827-4837, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31690231

RESUMO

The major threats linked to Zika virus (ZIKV) are microcephaly, Guillain-Barre syndrome, and the ability to transfer through sexual transmission. Despite these threats, Zika specific FDA approved drugs or vaccines are not available as of yet. Additionally, the involvement of pregnant women makes the drug screening process lengthy and complicated in terms of safety and minimum toxicity of the molecules. Since NS3 helicase of ZIKV performs the critical function of unwinding double-stranded RNA during replication, it is considered as a promising drug target to block ZIKV replication. In the present study, we have exploited the NTPase site of ZIKV NS3 helicase for screening potential inhibitor compounds by molecular docking, and molecular dynamics (MD) simulation approaches. NS3 helicase hydrolyzes the ATP to use its energy for unwinding RNA. We have chosen twenty natural compounds from ZINC library with known antiviral properties and a helicase focused library (HFL) of small molecules from Life Chemicals compounds. After going through docking, the top hit molecules from ZINC and HFL library were further analysed by MD simulations to find out stable binding poses. Finally, we have reported the molecules with potential of binding at NTPase pocket of ZIKV NS3 helicase, which could be further tested on virus through in vitro experiments to check their efficacy.Communicated by Ramaswamy H. Sarma.


Assuntos
Infecção por Zika virus , Zika virus , Feminino , Humanos , Simulação de Acoplamento Molecular , Nucleosídeo-Trifosfatase , Gravidez , RNA Helicases , Proteínas não Estruturais Virais
17.
Vet Parasitol ; 271: 38-44, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31303201

RESUMO

A nucleoside triphosphate diphosphohydrolase-1 (NTPDase 1) was identified on the surface, flagellum and kinetoplast from L. infantum promastigotes by immunocytochemistry and confocal laser scanning microscopy, using immune sera that recognized specifically the B domain of NTPDase 1 and produced against synthetic peptides (LbB1LJ and LbB2LJ) derived from this domain. The polyclonal antibodies had effective antileishmanial effect, reducing significantly in vitro promastigotes growth (21-25%), an antiproliferative effect also demonstrated by immune sera produced against recombinant r-pot B domain, and two other synthetic peptides (potB1LJ and potB2LJ). In addition, using these biomolecules in ELISA technique, IgG1 and IgG2 subclasses reactivities of either healthy dogs or infected by L. infantum and classified clinically as asymptomatic, oligosymptomatic and symptomatic were tested. Analysis of distinct IgG1 and IgG2 seropositivities patterns suggested antibody subclasses binding epitopes along B domain for protection against infection, indicating this domain as a new tool for prophylactic and immunotherapeutic investigations.


Assuntos
Anticorpos Antiprotozoários/imunologia , Doenças do Cão/imunologia , Imunoglobulina G/imunologia , Leishmania infantum/enzimologia , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Nucleosídeo-Trifosfatase/imunologia , Animais , Anticorpos Antiprotozoários/metabolismo , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Domínios Proteicos/imunologia
18.
Nucleic Acids Res ; 47(11): 5837-5851, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31066445

RESUMO

Ebola virus (EBOV) is a non-segmented, negative-sense RNA virus (NNSV) in the family Filoviridae, and is recognized as one of the most lethal pathogens in the planet. For RNA viruses, cellular or virus-encoded RNA helicases play pivotal roles in viral life cycles by remodelling viral RNA structures and/or unwinding viral dsRNA produced during replication. However, no helicase or helicase-like activity has ever been found to associate with any NNSV-encoded proteins, and it is unknown whether the replication of NNSVs requires the participation of any viral or cellular helicase. Here, we show that despite of containing no conserved NTPase/helicase motifs, EBOV VP35 possesses the NTPase and helicase-like activities that can hydrolyse all types of NTPs and unwind RNA helices in an NTP-dependent manner, respectively. Moreover, guanidine hydrochloride, an FDA-approved compound and inhibitor of certain viral helicases, inhibited the NTPase and helicase-like activities of VP35 as well as the replication/transcription of an EBOV minigenome replicon in cells, highlighting the importance of VP35 helicase-like activity during EBOV life cycle. Together, our findings provide the first demonstration of the NTPase/helicase-like activity encoded by EBOV, and would foster our understanding of EBOV and NNSVs.


Assuntos
Ebolavirus/genética , Ebolavirus/metabolismo , Doença pelo Vírus Ebola/virologia , Nucleoproteínas/fisiologia , RNA de Cadeia Dupla/química , Proteínas do Core Viral/fisiologia , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Células Cultivadas , DNA Helicases/metabolismo , Humanos , Proteínas do Nucleocapsídeo , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , RNA Helicases/metabolismo , RNA Viral/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
19.
Sci Rep ; 9(1): 7761, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123301

RESUMO

The ABC transporter Pdr5 of S. cerevisiae is a key player of the PDR network that works as a first line of defense against a wide range of xenobiotic compounds. As the first discovered member of the family of asymmetric PDR ABC transporters, extensive studies have been carried out to elucidate the molecular mechanism of drug efflux and the details of the catalytic cycle. Pdr5 turned out to be an excellent model system to study functional and structural characteristics of asymmetric, uncoupled ABC transporters. However, to date studies have been limited to in vivo or plasma membrane systems, as it was not possible to isolate Pdr5 in a functional state. Here, we describe the solubilization and purification of Pdr5 to homogeneity in a functional state as confirmed by in vitro assays. The ATPase deficient Pdr5 E1036Q mutant was used as a control and proves that detergent-purified wild-type Pdr5 is functional resembling in its activity the one in its physiological environment. Finally, we show that the isolated active Pdr5 is monomeric in solution. Taken together, our results described in this study will enable a variety of functional investigations on Pdr5 required to determine molecular mechanism of this asymmetric ABC transporter.


Assuntos
Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Transportadores de Cassetes de Ligação de ATP/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Nucleosídeo-Trifosfatase/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo
20.
PLoS Genet ; 15(5): e1008120, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116744

RESUMO

N6-Methyladenosine (m6A) RNA methylation plays important roles during development in different species. However, knowledge of m6A RNA methylation in monocots remains limited. In this study, we reported that OsFIP and OsMTA2 are the components of m6A RNA methyltransferase complex in rice and uncovered a previously unknown function of m6A RNA methylation in regulation of plant sporogenesis. Importantly, OsFIP is essential for rice male gametogenesis. Knocking out of OsFIP results in early degeneration of microspores at the vacuolated pollen stage and simultaneously causes abnormal meiosis in prophase I. We further analyzed the profile of rice m6A modification during sporogenesis in both WT and OsFIP loss-of-function plants, and identified a rice panicle specific m6A modification motif "UGWAMH". Interestingly, we found that OsFIP directly mediates the m6A methylation of a set of threonine protease and NTPase mRNAs and is essential for their expression and/or splicing, which in turn regulates the progress of sporogenesis. Our findings revealed for the first time that OsFIP plays an indispensable role in plant early sporogenesis. This study also provides evidence for the different functions of the m6A RNA methyltransferase complex between rice and Arabidopsis.


Assuntos
Gametogênese Vegetal , Regulação da Expressão Gênica de Plantas , Metiltransferases/genética , Oryza/genética , Proteínas de Plantas/genética , Subunidades Proteicas/genética , Adenosina/análogos & derivados , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Mutação com Perda de Função , Prófase Meiótica I , Metilação , Metiltransferases/metabolismo , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Subunidades Proteicas/metabolismo , RNA de Plantas , Especificidade da Espécie
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