RESUMO
Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here, we report the crystal structure of papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with the Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in a moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that 2-residue-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alphacoronavirus PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for the substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating (DUB) and proteolytic functions of PEDV PLP2 from a structural perspective. IMPORTANCE Current challenges in coronaviruses (CoVs) include a comprehensive understanding of the mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structural basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed that (i) three Ub core-interacting residues are essential for DUB function, (ii) 2-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only structural insights into the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.
Assuntos
Proteases Semelhantes à Papaína de Coronavírus/química , Vírus da Diarreia Epidêmica Suína/química , Coronavirus/química , Coronavirus/classificação , Coronavirus/enzimologia , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Cristalografia por Raios X , Mutação , Vírus da Diarreia Epidêmica Suína/enzimologia , Vírus da Diarreia Epidêmica Suína/genética , Ligação Proteica , Domínios Proteicos , Relação Estrutura-Atividade , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/metabolismoRESUMO
Papain-like protease (PLpro) is one of two cysteine proteases involved in the proteolytic processing of the polyproteins of Severe acute respiratory syndrome coronavirus (SARS-CoV). PLpro also shows significant in vitro deubiquitinating and de-ISGylating activities, although the detailed mechanism is still unclear. Here, the crystal structure of SARS-CoV PLpro C112S mutant in complex with ubiquitin (Ub) is reported at 1.4â Å resolution. The Ub core makes mostly hydrophilic interactions with PLpro, while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PLpro, mimicking the P4-P1 residues and providing the first atomic insights into its catalysis. One of the O atoms of the C-terminal Gly residue of Ub is located in the oxyanion hole consisting of the main-chain amides of residues 112 and 113. Mutations of residues in the PLpro-Ub interface lead to reduced catalytic activity, confirming their importance for Ub binding and/or catalysis. The structure also revealed an N-cyclohexyl-2-aminethanesulfonic acid molecule near the catalytic triad, and kinetic studies suggest that this binding site is also used by other PLpro inhibitors. Overall, the structure provides a foundation for understanding the molecular basis of coronaviral PLpro catalysis.
Assuntos
Papaína/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Ubiquitina/química , Proteínas Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Papaína/genética , Papaína/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteólise , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Taurina/análogos & derivados , Taurina/química , Taurina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
BACKGROUNDS: A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated. RESULTS: Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference. CONCLUSIONS: Overall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Proteínas Virais/química , Proteínas Virais/genética , Antivirais/química , Proteases 3C de Coronavírus , Cisteína Endopeptidases/biossíntese , Europa (Continente) , Regulação Viral da Expressão Gênica , Humanos , Íons/química , Metais/química , Processamento de Proteína Pós-Traducional , Proteólise , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas Virais/biossínteseRESUMO
The Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti-SARS drugs. The functional unit of M(pro) is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on M(pro); nevertheless, the mechanism by which monomeric M(pro) is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C-terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate-binding pocket. Interestingly, the R298A mutant of M(pro) shows a reversible substrate-induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate-induced dimerization is delineated by X-ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild-type protease is present in the asymmetric unit. The presence of a complete substrate-binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate-induced dimerization and has important implications with respect to the maturation of the enzyme.
Assuntos
Cisteína Endopeptidases/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas da Matriz Viral/química , Proteínas Virais/química , Sítios de Ligação , Proteases 3C de Coronavírus , Proteínas M de Coronavírus , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Multimerização Proteica , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Papain-like protease (PLpro) from severe acute respiratory syndrome (SARS) coronavirus is one of the two proteases involved in the proteolytic processing of the virion polyproteins. In addition, PLpro shows significant in vitro deubiquitinating and de-ISGylating activities. All these findings demonstrated the multifunctional nature of the PLpro. Here we report the sensitivity of PLpro to denaturant urea. An increase in urea concentration induced a reversible biphasic unfolding of the enzyme. Differently, the unfolding of the catalytic triad region located within the palm and thumb domains followed a monophasic unfolding curve. Further observations suggest that the zinc-binding domain may start to unfold during the first transition. An 80% lost of its enzymatic activity at a urea concentration lower than 1M showed a close correlation with unfolding of the zinc-binding domain. The enzyme was also characterized in terms of hydrophobicity and size-and-shape distribution. We have demonstrated that PLpro displayed differential domain structure stability and molten globule state in its folding. These studies will not only assist in our understanding of the folding of this viral enzyme, but also that of other deubiquitinating enzymes with a similar scaffold.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/ultraestrutura , Modelos Químicos , Modelos Moleculares , Ureia/química , Proteínas Virais/química , Proteínas Virais/ultraestrutura , Sequência de Aminoácidos , Proteases 3C de Coronavírus , Ativação Enzimática , Estabilidade Enzimática , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Purpose: To develop a zebrafish cataract model for screening potential anti-cataract compounds. Methods: Living zebrafish were anesthetized and exposed to ultraviolet-C (UV-C) irradiation at a dosage of 3250 mJ/cm2/d until they developed severe cataracts. These cataracts were graded based on photographs analyzed with ImageQuant TL version 7.0. Fish with severe cataracts were used to evaluate a range of compounds for cataract treatment, including the previously demonstrated hit compound lanosterol. For the initial evaluation, fish were divided into four groups: no treatment, balanced salt solution, ß-cyclodextrin (ß-CD), and lanosterol dissolved in ß-CD. The treatments were performed for 10 days, and the clarity of lenses was evaluated. To assess the persistence of treatment, fish were treated with ß-CD and lanosterol dissolved in ß-CD for seven consecutive days followed by monitoring for three days without treatment. Results: The average time for zebrafish to develop severe cataracts using the present UV-C irradiation protocol was 7.8 days (range 4-15 days). Both study designs required only another 10 days to determine the effect of hit compounds. The total experimental period could be completed within one month, and the entire experiment was economical. Conclusions: We could assay a large number of hit compounds at a reasonable cost and within a short time using this newly developed zebrafish cataract model. These assays may allow development of an efficient platform for screening potential anti-cataract compounds. Translational Relevance: The results may facilitate the development of ani-cataract medication for humans after further experiments and investigations.
Assuntos
Catarata , Cristalino , Animais , Catarata/tratamento farmacológico , Modelos Animais de Doenças , Humanos , Lanosterol/farmacologia , Lanosterol/uso terapêutico , Cristalino/efeitos da radiação , Peixe-ZebraRESUMO
Capsulin is one of the transcription factors involved in regulating cell differentiation but its biochemical properties and structural characteristics are still unclear. In the present study, we cloned capsulin from zebrafish, which produces large numbers of transparent embryos and has well-characterized developmental stages. By alignment, the deduced amino acid sequence of zebrafish Capsulin, which contains a putative bHLH motif, shares very high homology to that of other species with an 72-82% identity. Zebrafish Capsulin was also targeted to the nucleus of mammalian cells when overexpressed by transient transfection. In order to characterize the structural and biochemical properties of zebrafish Capsulin, a recombinant zebrafish Capsulin protein was expressed and purified in Escherichia coli. By circular dichroism spectroscopy, Capsulin was shown to be 55% α-helical. The size distribution assay by analytical ultracentrifugation indicated that it existed as a monomer-dimer mixture. The results suggested that the recombinant Capsulin has a well-organized and functional structure. Finally, endogenous Capsulin was distributed mainly in the epicardial cells of zebrafish by immunohistochemistry analysis using antibodies raised against zebrafish Capsulin. The present study not only helps us to comparatively analyze capsulin genes across species, but it also provides valuable structural information for further studies of Capsulin biological function in the future.
Assuntos
Escherichia coli/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/análise , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Linhagem Celular , Núcleo Celular/ultraestrutura , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Humanos , Dados de Sequência Molecular , Pericárdio/citologia , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Transfecção , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/isolamento & purificaçãoRESUMO
Disulfiram is a promising repurposed drug that, combining with radiation and chemotherapy, exhibits effective anticancer activities in several preclinical models. The cellular metabolites of disulfiram have been established, however, the intracellular targets of disulfiram remain largely unexplored. We have previously reported that disulfiram suppresses the coronaviral papain-like proteases through attacking their zinc-finger domains, suggesting an inhibitory function potentially on other proteases with similar catalytic structures. Ubiquitin-specific proteases (USPs) share a highly-conserved zinc-finger subdomain that structurally similar to the papain-like proteases and are attractive anticancer targets as upregulated USPs levels are found in a variety of tumors. Here, we report that disulfiram functions as a competitive inhibitor for both USP2 and USP21, two tumor-related deubiquitinases. In addition, we also observed a synergistic inhibition of USP2 and USP21 by disulfiram and 6-Thioguanine (6TG), a clinical drug for acute myeloid leukemia. Kinetic analyses revealed that both drugs exhibited a slow-binding mechanism, moderate inhibitory parameters, and a synergistically inhibitory effect on USP2 and USP21, suggesting the potential combinatory use of these two drugs for USPs-related tumors. Taken together, our study provides biochemical evidence for repurposing disulfiram and 6TG as a combinatory treatment in clinical applications.
Assuntos
Dissulfiram/química , Inibidores Enzimáticos/química , Tioguanina/química , Ubiquitina Tiolesterase , Dissulfiram/agonistas , Sinergismo Farmacológico , Humanos , Tioguanina/agonistas , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/químicaRESUMO
The maturation of SARS coronavirus involves the autocleavage of polyproteins 1a and 1ab by the main protease (Mpro) and a papain-like protease; these represent attractive targets for the development of anti-SARS drugs. The functional unit of Mpro is a homodimer, and each subunit has a His-41cdots, three dots, centeredCys-145 catalytic dyad. Current thinking in this area is that Mpro dimerization is essential for catalysis, although the influence of the substrate binding on the dimer formation has never been explored. Here, we delineate the contributions of the peptide substrate to Mpro dimerization. Enzyme kinetic assays indicate that the monomeric mutant R298A/L exhibits lower activity but in a cooperative manner. Analytical ultracentrifugation analyses indicate that in the presence of substrates, the major species of R298A/L shows a significant size shift toward the dimeric form and the monomer-dimer dissociation constant of R298A/L decreases by 12- to 17-fold, approaching that for wild-type. Furthermore, this substrate-induced dimerization was found to be reversible after substrates were removed. Based on the crystal structures, a key residue, Glu-166, which is responsible for recognizing the Gln-P1 of the substrate and binding to Ser-1 of another protomer, will interact with Asn-142 and block the S1 subsite entrance in the monomer. Our studies indicate that mutation of Glu-166 in the R298A mutant indeed blocks the substrate-induced dimerization. This demonstrates that Glu-166 plays a pivotal role in connecting the substrate binding site with the dimer interface. We conclude that protein-ligand and protein-protein interactions are closely correlated in Mpro.
Assuntos
Cisteína Endopeptidases/química , Ácido Glutâmico/química , Mutação , Proteínas Virais/química , Área Sob a Curva , Sítios de Ligação , Catálise , Cromatografia/métodos , Proteases 3C de Coronavírus , Cristalografia por Raios X/métodos , Óxido de Deutério/química , Dimerização , Cinética , Peptídeos/química , Ultracentrifugação , Água/químicaRESUMO
Diabetes mellitus together with the oxidative stress affects the process of spermatogenesis and leads to male infertility. Antrodia cinnamomea (AC) is a mushroom found unique in Taiwan and commonly used for the treatment of several types of cancers and inflammatory disorders. This study was aimed to investigate the anti-oxidative and the ameliorative effects of Antrodia cinnamomea ethanol extract (ACEE) on reproduction dysfunction in male diabetic rats. The diabetic condition was induced by administrating the combination of streptozotocin (STZ) (65 mg/kg) and nicotinamide (NA) (230 mg/kg). Three different doses of ACEE were tested (385, 770, 1540 mg/kg) for 5 weeks. The results indicated that the ACEE improved STZ-NA induced hyperglycemia, oxidative stress, and insulin resistance. In addition to this, ACEE reduced the degree of lipid peroxidation, recovered the abnormal structure of the seminiferous tubules, and improved sperm parameters. Moreover, the DNA damages and mitochondrial membrane potential were improved in sperm. Our study confirmed that the ACEE has anti-inflammatory and ameliorative effects to prevent diabetes-induced male reproductive dysfunction.
Assuntos
Antrodia , Diabetes Mellitus Experimental/tratamento farmacológico , Etanol/administração & dosagem , Extratos Vegetais/uso terapêutico , Reprodução/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Relação Dose-Resposta a Droga , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Masculino , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Ratos , Ratos Sprague-Dawley , Reprodução/fisiologia , Motilidade dos Espermatozoides/fisiologiaRESUMO
The performance of an analytical expression for algorithmic decoherence time is investigated for non-Born-Oppenheimer molecular dynamics. There are two terms in the function that represents the dependence of the decoherence time on the system parameters; one represents decoherence due to the quantum time-energy uncertainty principle and the other represents a back reaction from the decoherent force on the classical trajectory. We particularly examine the question of whether the first term should dominate. Five one-dimensional two-state model systems that represent limits of multidimensional nonadiabatic dynamics are designed for testing mixed quantum-classical methods and for comparing semiclassical calculations with exact quantum calculations. Simulations are carried out with the semiclassical Ehrenfest method (SE), Tully's fewest switch version (TFS) of the trajectory surface hopping method, and the decay-of-mixing method with natural switching, coherent switching (CSDM), and coherent switching with reinitiation (CSDM-D). The CSDM method is demonstrated to be the most accurate method, and it has several desirable features: (i) It behaves like the representation-independent SE method in the strong nonadiabatic coupling regions; (ii) it behaves physically like the TFS method in noninteractive region; and (iii) the trajectories are continuous with continuous momenta. The CSDM method is also demonstrated to balance coherence well with decoherence, and the results are nearly independent of whether one uses the adiabatic or diabatic representation. The present results provide new insight into the formulation of a physically correct decoherence time to be used with the CSDM method for non-Born-Oppenheimer molecular dynamic simulations.
RESUMO
Diabetes is a chronic disorder characterized by hyperglycemia due to decreased levels of insulin or the inefficiency of the tissue to use it effectively. Infertility is known as a major outcome of diabetes and affects the male reproductive system by causing sperm impairment and gonadal dysfunction. Cistanche tubulosa is a parasitic plant which has the capacity to improve memory, immunity, and sexual ability, reduce impotence, and minimize constipation. This study was focused on the investigation of the anti-inflammatory and protective effects of echinacoside (ECH) in Cistanche tubulosa extract (CTE) on the male reproductive system of the diabetic rats. The antioxidant, anti-inflammatory, and protective effects of CTE were evaluated by both in vitro and in vivo methods. The in vitro results show that the ECH inhibited reactive oxygen species (ROS) production and improved StAR, CYP11A1, CYP17A1, and HSD17ß3 protein expression. The in vivo analysis was carried out with three doses of echinacoside (ECH) (80, 160, and 320 mg/kg) in CTE. In total, 0.571 mg/kg of rosiglitazone (RSG) was administered as a positive control. Diabetes was induced by streptozotocin (STZ) (65 mg/kg) and nicotinamide (230 mg/kg) in combination with a high-fat diet (45%). The in vivo studies confirmed that the ECH improved blood sugar levels, insulin resistance, leptin resistance, and lipid peroxidation. It can restore kisspeptin 1 (KiSS1), G protein-coupled receptor GPR 54, suppressor of cytokine signaling 3 (SOCS-3), and sirtuin 1 (SIRT1) messenger ribonucleic acid (mRNA) expression in the hypothalamus and recover sex hormone level. Thus, this study confirmed the antioxidant, anti-inflammatory, and steroidogenesis effects of CTE.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Cistanche/química , Diabetes Mellitus Experimental/induzido quimicamente , Extratos Vegetais/farmacologia , Testículo/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Antioxidantes/química , Glicemia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colesterol/sangue , Diabetes Mellitus Experimental/complicações , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase , Peróxido de Hidrogênio/metabolismo , Insulina/sangue , Leptina/sangue , Hormônio Luteinizante/sangue , Masculino , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Testículo/metabolismo , Testosterona/sangue , Triglicerídeos/sangueRESUMO
Ubiquitin-specific protease 2 (USP2) belongs to the family of deubiquitinases that can rescue protein targets from proteasomal degradation by reversing their ubiquitination. In various cancers, including prostate cancer and ovarian carcinoma, upregulation of USP2 leads to an increase in the levels of deubiquitinated substrates such as fatty acid synthase, MDM2, cyclin D1 and Aurora-A. USP2 thus plays a critical role in tumor cells' survival and therefore represents a therapeutic target. Here a leukemia drug, 6-thioguanine, was found to be a potent inhibitor of USP2. Enzyme-kinetic and X-ray crystallographic data suggest that 6-thioguanine displays a noncompetitive and slow-binding inhibitory mechanism against USP2. Our study provides a clear rationale for the clinical evaluation of 6-thioguanine for USP2-upregulated cancers.
Assuntos
Endopeptidases/metabolismo , Tioguanina/farmacologia , Cristalografia por Raios X/métodos , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/metabolismo , Humanos , Cinética , Processamento de Proteína Pós-Traducional , Tioguanina/metabolismo , Tioguanina/farmacocinética , Ubiquitina Tiolesterase , Ubiquitinação/fisiologiaRESUMO
Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in southern China in late 2002 and caused a global outbreak with a fatality rate around 10% in 2003. Ten years later, a second highly pathogenic human CoV, MERS-CoV, emerged in the Middle East and has spread to other countries in Europe, North Africa, North America and Asia. As of November 2017, MERS-CoV had infected at least 2102 people with a fatality rate of about 35% globally, and hence there is an urgent need to identify antiviral drugs that are active against MERS-CoV. Here we show that a clinically available alcohol-aversive drug, disulfiram, can inhibit the papain-like proteases (PLpros) of MERS-CoV and SARS-CoV. Our findings suggest that disulfiram acts as an allosteric inhibitor of MERS-CoV PLpro but as a competitive (or mixed) inhibitor of SARS-CoV PLpro. The phenomenon of slow-binding inhibition and the irrecoverability of enzyme activity after removing unbound disulfiram indicate covalent inactivation of SARS-CoV PLpro by disulfiram, while synergistic inhibition of MERS-CoV PLpro by disulfiram and 6-thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs.
Assuntos
Antivirais/farmacologia , Dissulfiram/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Peptídeo Hidrolases/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Dissulfiram/química , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Modelos Moleculares , Conformação Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Ligação Proteica , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genéticaRESUMO
Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We've also identified four historical mutations that are able to produce a "GST-like" S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution.
Assuntos
Cristalinas , Evolução Molecular , Glutationa Transferase , Mutação , Octopodiformes , Animais , Cristalinas/química , Cristalinas/genética , Cristalografia por Raios X , Glutationa Transferase/química , Glutationa Transferase/genética , Octopodiformes/química , Octopodiformes/genética , Domínios ProteicosRESUMO
BACKGROUND: A highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and other places in Saudi Arabia, and has quickly spread to European and Asian countries since September 2012. Up to the 1st October 2015 it has infected at least 1593 people with a global fatality rate of about 35%. Studies to understand the virus are necessary and urgent. In the present study, MERS-CoV main protease (Mpro) is expressed; the dimerization of the protein and its relationship to catalysis are investigated. METHODS AND RESULTS: The crystal structure of MERS-CoV Mpro indicates that it shares a similar scaffold to that of other coronaviral Mpro and consists of chymotrypsin-like domains I and II and a helical domain III of five helices. Analytical ultracentrifugation analysis demonstrated that MERS-CoV Mpro undergoes a monomer to dimer conversion in the presence of a peptide substrate. Glu169 is a key residue and plays a dual role in both dimerization and catalysis. The mutagenesis of other residues found on the dimerization interface indicate that dimerization of MERS-CoV Mpro is required for its catalytic activity. One mutation, M298R, resulted in a stable dimer with a higher level of proteolytic activity than the wild-type enzyme. CONCLUSIONS: MERS-CoV Mpro shows substrate-induced dimerization and potent proteolytic activity. A critical assessment of the residues important to these processes provides insights into the correlation between dimerization and catalysis within the coronaviral Mpro family.
Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Peptídeo Hidrolases/química , Proteínas Virais/química , Cristalografia por Raios X , Modelos Moleculares , Papaína/química , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , Multimerização Proteica , Análise de Sequência de Proteína , Proteínas Virais/análiseRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) is a new highly pathogenic human coronaviruses that emerged in Jeddah and Saudi Arabia and has quickly spread to other countries in Middle East, Europe and North Africa since 2012. Up to 17 December 2014, it has infected at least 938 people with a fatality rate of about 36% globally. This has resulted in an urgent need to identify antiviral drugs that are active against MERS-CoV. The papain-like protease (PL(pro)) of MERS-CoV represents an important antiviral target as it is not only essential for viral maturation, but also antagonizes interferon stimulation of the host via its deubiquitination activity. Here, we report the discovery that two SARS-CoV PL(pro) inhibitors, 6-mercaptopurine (6MP) and 6-thioguanine (6TG), as well as the immunosuppressive drug mycophenolic acid, are able to inhibit MERS-CoV PL(pro). Their inhibition mechanisms and mutually binding synergistic effect were also investigated. Our results identify for the first time three inhibitors targeting MERS-CoV PL(pro) and these can now be used as lead compounds for further antiviral drug development.
Assuntos
Antivirais/farmacologia , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Mercaptopurina/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Ácido Micofenólico/farmacologia , Tioguanina/farmacologia , Proteases 3C de Coronavírus , Cisteína Endopeptidases , Cisteína Proteases/química , Sinergismo Farmacológico , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Modelos Moleculares , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas Virais/antagonistas & inibidoresRESUMO
Here, we investigated whether aminophylline, an adenosine receptor antagonist used usually as a treatment for premature apnea, had synergistic effects on status epilepticus in the developing brain. On postnatal day 14 (P14), four groups of rats intraperitoneally received saline, aminophylline, lithium--pilocarpine (Li-PC), and Li-PC plus aminophylline, respectively. Subsequently, the Morris water maze task was performed at P80. The brains were then analyzed with cresyl violet stain for histological lesions and evaluated for mossy fiber sprouting with the Timm stain. No seizures were elicited in the saline-treated or aminophylline-treated rats. Both the Li-PC-treated and aminophylline plus Li-PC-treated rats exhibited seizures and there was no significant difference in mortality between the two groups. More interestingly, as in adulthood (P80), aminophylline aggravated the spatial deficits and histological damages seen in Li-PC-treated rats. In summary, this present study suggests that the use of adenosine receptor antagonists, such as aminophylline, exacerbates seizure-induced damage in the developing brain.
Assuntos
Aminofilina/toxicidade , Transtornos Cognitivos/fisiopatologia , Hipocampo/crescimento & desenvolvimento , Degeneração Neural/fisiopatologia , Inibidores de Fosfodiesterase/toxicidade , Antagonistas de Receptores Purinérgicos P1 , Estado Epiléptico/fisiopatologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/patologia , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/patologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Compostos de Lítio/farmacologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Fibras Musgosas Hipocampais/efeitos dos fármacos , Fibras Musgosas Hipocampais/patologia , Agonistas Muscarínicos/farmacologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Pilocarpina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P1/metabolismo , Convulsões/induzido quimicamente , Convulsões/patologia , Convulsões/fisiopatologia , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/patologiaRESUMO
The Ca-exchanged clays were used to investigate the sorption of toluene under both high and low relative humidity conditions. According to the observed rates of sorption and desorption and the Fourier transform infrared (FTIR) spectra of sorbed toluene, the sorbate molecules on clays can be divided into three categories: labile (gaslike) phase, slow sorption (liquidlike) phase, and resistant-to-desorption phase with sorption/desorption time scales of less than 30 min, several hours, and several weeks, respectively. Aging changed the spectroscopic characteristics of the sorbed sorbates and contributed to the irreversibility of the sorption under both high and low relative humidity. Irreversibly sorbed portions of the sorbates and new characteristic peaks identified with FTIR spectrometry suggest the existence of chemical transformation of sorbed species under both high and low relative humidity conditions. It is suggested that the clay minerals, acting as catalysts, may contribute to abiotic natural attenuation of volatile organic aromatic hydrocarbons in soils.