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1.
Front Immunol ; 13: 883079, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35479093

RESUMO

Mammalian neuraminidases (NEUs), also known as sialidases, are enzymes that cleave off the terminal neuraminic, or sialic, acid resides from the carbohydrate moieties of glycolipids and glycoproteins. A rapidly growing body of literature indicates that in addition to their metabolic functions, NEUs also regulate the activity of their glycoprotein targets. The simple post-translational modification of NEU protein targets-removal of the highly electronegative sialic acid-affects protein folding, alters protein interactions with their ligands, and exposes or covers proteolytic sites. Through such effects, NEUs regulate the downstream processes in which their glycoprotein targets participate. A major target of desialylation by NEUs are mucins (MUCs), and such post-translational modification contributes to regulation of disease processes. In this review, we focus on the regulatory roles of NEU-modified MUCs as coordinators of disease pathogenesis in fibrotic, inflammatory, infectious, and autoimmune diseases. Special attention is placed on the most abundant and best studied NEU1, and its recently discovered important target, mucin-1 (MUC1). The role of the NEU1 - MUC1 axis in disease pathogenesis is discussed, along with regulatory contributions from other MUCs and other pathophysiologically important NEU targets.


Assuntos
Doenças do Sistema Imunitário , Mucinas , Animais , Glicoproteínas/metabolismo , Mamíferos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo
2.
Oncogene ; 41(18): 2638-2650, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35354905

RESUMO

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients.


Assuntos
Sarcoma de Ewing , Animais , Linhagem Celular Tumoral , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histona Metiltransferases/metabolismo , Humanos , Camundongos , Recidiva Local de Neoplasia , Neuraminidase/metabolismo , Neuraminidase/uso terapêutico , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia
3.
J Proteomics ; 259: 104544, 2022 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-35240312

RESUMO

Madin-Darby canine kidney (MDCK) cells are widely used in basic research and for the propagation of influenza A viruses (IAV) for vaccine production. To identify targets for antiviral therapies and to optimize vaccine manufacturing, a detailed understanding of the viral life cycle is important. This includes the characterization of virus entry, the synthesis of the various viral RNAs and proteins, the transfer of viral compounds in the cell and virus budding. In case quantitative information is available, the analysis can be complemented by mathematical modelling approaches. While comprehensive studies focusing on IAV entry as well as viral mRNA, vRNA and cRNA accumulation in the nucleus of cells have been performed, quantitative data regarding IAV protein synthesis and accumulation was mostly lacking. In this study, we present a mass spectrometry (MS)-based method to evaluate whether an absolute quantification of viral proteins is possible for single-round replication in suspension MDCK cells. Using influenza A/PR/8/34 (H1N1, RKI) as a model strain at a multiplicity of infection of ten, defined amounts of isotopically labelled peptides of synthetic origin of four IAV proteins (hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1) were added as an internal standard before tryptic digestion of samples for absolute quantification (AQUA). The first intracellular protein detected was NP at 1 h post infection (hpi). A maximum extracellular concentration of 7.7E+12 copies/mL was achieved. This was followed by hemagglutinin (3 hpi, maximum 4.1E+12 copies/mL at 13 hpi), matrix protein 1 (5 hpi, maximum 2.2E+12 copies/mL at 13 hpi) and neuraminidase (5 hpi, 6.0E+11 copies/mL at 13 hpi). In sum, for the first time absolute IAV protein copy numbers were quantified by a MS-based method for infected MDCK cells providing important insights into viral protein dynamics during single-round virus replication. SIGNIFICANCE: Influenza A virus is a significant human pathogen worldwide. To improve therapies against influenza and overcome bottlenecks in vaccine production in cell culture, it is critical to gain a detailed understanding of the viral life cycle. In addition to qPCR-based models, this study will examine the dynamics of influenza virus proteins during infection of producer cells to gain initial insights into changes in absolute copy numbers.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Animais , Cães , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Células Madin Darby de Rim Canino , Neuraminidase/genética , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Replicação Viral
4.
Comput Biol Med ; 144: 105290, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35231801

RESUMO

Neuraminidase A from Streptococcus pneumoniae (NanA) is considered a potentially key pathogenicity factor and a promising drug target to treat human infectious diseases. Computational and experimental efforts are increasingly being used to study its structure and function which yet remain poorly understood. In this work, we characterized structural dynamics of NanA's active site and gained novel mechanistic insights into its implications for a ligand binding. We based our study on supercomputer modeling and bioinformatic analysis with a help of crystallographic data and by bringing together previously published experimental data. The most prominent conformational plasticity was observed in the loop 422-437, accompanied by the mobility of adjacent loops 352-360 and 579-587. These structural elements had been undergoing spontaneous fluctuations apparently playing the role of an active site lid: an "open" state allowed substrate access to the active site, while a "closed" state accommodated the substrate in a catalytically favorable orientation. We observed that conformational plasticity of the loop 422-437 promoted the formation of an additional pocket located between catalytic and insertion domains of the enzyme. We recently argued this site was able to bind isoprenylated flavone artocarpin as an inhibitor of pneumococcal biofilm formation. Here we showed that accommodation of the mixed-type inhibitor artocarpin in this pocket limited mobility of the loop 422-437. This represents a plausible explanation of artocarpin's regulatory effect on the enzyme's catalytic function which seems to be independent of its role in preventing biofilm formation.


Assuntos
Neuraminidase , Streptococcus pneumoniae , Regulação Alostérica , Domínio Catalítico , Humanos , Neuraminidase/química , Neuraminidase/metabolismo , Streptococcus pneumoniae/fisiologia
5.
Biochim Biophys Acta Mol Basis Dis ; 1868(5): 166368, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35202791

RESUMO

AIMS: Identifying the mechanisms that underlie progression from endothelial damage to podocyte damage, which leads to massive proteinuria, is an urgent issue that must be clarified to improve renal outcome in diabetic kidney disease (DKD). We aimed to examine the role of dynamin-related protein 1 (Drp1)-mediated regulation of mitochondrial fission in podocytes in the pathogenesis of massive proteinuria in DKD. METHODS: Diabetes- or albuminuria-associated changes in mitochondrial morphology in podocytes were examined by electron microscopy. The effects of albumin and other diabetes-related stimuli, including high glucose (HG), on mitochondrial morphology were examined in cultured podocytes. The role of Drp1 in podocyte damage was examined using diabetic podocyte-specific Drp1-deficient mice treated with neuraminidase, which removes endothelial glycocalyx. RESULTS: Neuraminidase-induced removal of glomerular endothelial glycocalyx in nondiabetic mice led to microalbuminuria without podocyte damage, accompanied by reduced Drp1 expression and mitochondrial elongation in podocytes. In contrast, streptozotocin-induced diabetes significantly exacerbated neuraminidase-induced podocyte damage and albuminuria, and was accompanied by increased Drp1 expression and enhanced mitochondrial fission in podocytes. Cell culture experiments showed that albumin stimulation decreased Drp1 expression and elongated mitochondria, although HG inhibited albumin-associated changes in mitochondrial dynamics, resulting in apoptosis. Podocyte-specific Drp1-deficiency in mice prevented diabetes-related exacerbation of podocyte damage and neuraminidase-induced development of albuminuria. Endothelial dysfunction-induced albumin exposure is cytotoxic to podocytes. Inhibition of mitochondrial fission in podocytes is a cytoprotective mechanism against albumin stimulation, which is impaired under diabetic condition. Inhibition of mitochondrial fission in podocytes may represent a new therapeutic strategy for massive proteinuria in DKD.


Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Podócitos , Albuminas/metabolismo , Albuminas/farmacologia , Albuminúria/genética , Albuminúria/metabolismo , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/patologia , Feminino , Humanos , Masculino , Camundongos , Dinâmica Mitocondrial , Neuraminidase/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Proteinúria/patologia
6.
Biomolecules ; 12(2)2022 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-35204831

RESUMO

Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the H1N1 influenza virus produced this overexpressed enzyme in the insoluble fraction of cells in the form of inclusion bodies. The aim of this work was to investigate the effect of independent variables (propagation time, isopropyl ß-d-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). The maximum yield of NA (112.97 ± 2.82 U/g) was achieved under optimal conditions, namely, a propagation time of 7.72 h, IPTG concentration of 1.82 mM and gene expression time of 7.35 h. This study demonstrated that bacterially expressed NA was enzymatically active.


Assuntos
Escherichia coli , Corpos de Inclusão , Vírus da Influenza A Subtipo H1N1 , Neuraminidase , Isopropiltiogalactosídeo/genética , Isopropiltiogalactosídeo/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo
7.
Vet Microbiol ; 266: 109371, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35176607

RESUMO

Necrotic enteritis, caused by NetB producing Clostridium perfringens type G strains, is a globally important poultry disease. An initial step in the pathogenesis of necrotic enteritis is the colonization and degradation of the intestinal mucus layer, a process in which C. perfringens sialidases - such as NanI sialidase - may play an important role. Sialidases cleave terminal sialic acid from complex carbohydrates on glycoconjugates, such as mucins. This study shows that NE-associated C. perfringens strain CP56 is able to use sialic acid (Neu5Ac) as a carbon source for bacterial growth. It is shown that supplementation of Neu5Ac in the growth medium does not only induce the production of extracellular sialidases of strain CP56, but also increases the production of both alpha toxin and NetB toxin. Moreover, it was found that pre-treating avian hepatocellular carcinoma cells (LMH cells) with the recombinant NanI sialidase increases the adherence of C. perfringens type G strain CP56 to these cells. As such, the data suggest an important role for sialidases in the pathogenesis of the disease.


Assuntos
Infecções por Clostridium , Clostridium perfringens , Animais , Infecções por Clostridium/veterinária , Clostridium perfringens/enzimologia , Clostridium perfringens/patogenicidade , Enterite/veterinária , Técnicas In Vitro , Intestinos/microbiologia , Mucinas/metabolismo , Neuraminidase/metabolismo
8.
Sci Rep ; 12(1): 3216, 2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35217678

RESUMO

Sialic acid (SA) is present in glycoconjugates and important in cell-cell recognition, cell adhesion, and cell growth and as a receptor. Among the four mammalian sialidases, cytosolic NEU2 has a pivotal role in muscle and neuronal differentiation in vitro. However, its biological functions in vivo remain unclear due to its very low expression in humans. However, the presence of cytoplasmic glycoproteins, gangliosides, and lectins involved in cellular metabolism and glycan recognition has suggested the functional importance of cytosolic Neu2 sialidases. We generated a Neu2 knockout mouse model via CRISPR/Cas9-mediated genome engineering and analyzed the offspring littermates at different ages to investigate the in vivo function of cytosolic Neu2 sialidase. Surprisingly, knocking out the Neu2 gene in vivo abrogated overall lipid metabolism, impairing motor function and leading to diabetes. Consistent with these results, Neu2 knockout led to alterations in sialylated glycoproteins involved in lipid metabolism and muscle function, as shown by glycoproteomics analysis.


Assuntos
Metabolismo dos Lipídeos , Músculos , Neuraminidase , Animais , Citosol/metabolismo , Mamíferos/metabolismo , Camundongos , Músculos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo
9.
Biomed Khim ; 68(1): 7-17, 2022 Jan.
Artigo em Russo | MEDLINE | ID: mdl-35221292

RESUMO

Sialic acids (SA) are derivatives of neuraminic acid; they are located at the terminal position in the chains of monosaccharide residues of various glycoconjugates. SA play a dual role, they either mask recognition sites, or, on the contrary, represent biological targets that can be recognized by receptor proteins and serve as ligands. The desialylation/sialylation processes can be viewed as a dynamic modification regulated by sialyltransferases and sialidases in response to external or internal stimuli. This review describes the structural and functional diversity and the potential use of SA fractions as biomarkers for various pathological conditions. Almost any extreme effects on the body and inflammatory processes lead to an increase in the level of both total and free SA in the blood and tissues. Possible reasons for the increase of sialoglycoconjugate metabolism indicators in biological material include activation of the hepatocyte synthesis and secretion of various acute-phase proteins, many of which are sialoglycoproteins, violation of the membrane integrity and destruction of body cells, and also high activity of sialidases (neurominidases) and sialyltransferases. Most acute and chronic liver diseases are characterized by the decrease in the total level of SA in the blood serum (because many plasma proteins are synthesized and glycosylated in hepatocytes). Aberrant sialylation results in changes of sialoglycoconjugate structure, its ability to perform biological functions and half-life. Glycosylation is the most common post-translational modification of proteins in the virus, which not only promotes the formation of specific conformation of viral proteins, but also modulates their interaction with receptors and affects host cell recognition, viral replication and infectivity. Serum total SA concentration increases in some benign and inflammatory conditions, which indicates a lack of specificity and limits their use for early detection and screening of neoplastic diseases. Nevertheless, determining blood SA level and measuring concentration of existing biomarkers can be used to improve diagnostic indicators, to stage and monitor therapeutic response in some types of cancer, when the need for specificity is less than for diagnosis. Clinical and diagnostic value of determining the sialoglycoconjugate metabolic indicators, including changes in the content of both SA fractions and specific proteins in various biological fluids and tissues, lies in establishing the causes and mechanisms of biochemical changes in the body in certain diseases.


Assuntos
Neoplasias , Ácidos Siálicos , Glicoconjugados , Humanos , Neoplasias/metabolismo , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo
10.
Bioorg Med Chem ; 57: 116647, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35121400

RESUMO

Neuraminidase (NA) is an important target in the development of anti-influenza virus drugs. Compounds containing 1,3, 4-oxadiazole heterocycles have good biological activity and have been proved to have wide applications in antibacterial and antiviral drugs. In this paper, a series of novel 1, 3, 4-oxadiazole neuraminidase inhibitors (6a-6l) were designed and synthesized and their inhibitory activities of NA was tested in vitro. The results displayed that compound 6d exerts the best inhibitory activity (IC50 = 0.027 µM), which was obviously lower than that of oseltamivir carboxylate (OSC) (IC50 = 0.082 µM). Molecular docking analysis showed that the 1, 3, 4-oxadiazole heterocycle plays crucial part in compound 6d, and it can interact with the key arginine triad (Arg118, Arg292 and Arg 371) at the NA S1 site. The good efficacy of 6d may also be attributed to the extension of the substituted aniline ring to the 150-cavitiy. The theoretical and experimental results may provide reference for development of new anti-influenza drugs.


Assuntos
Antivirais/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Orthomyxoviridae/efeitos dos fármacos , Oxidiazóis/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Neuraminidase/metabolismo , Oxidiazóis/síntese química , Oxidiazóis/química , Relação Estrutura-Atividade
11.
Bioorg Med Chem Lett ; 61: 128590, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35108623

RESUMO

Neuraminidase (NA) is an ideal target for the development of anti-influenza drugs. In this paper, ZINC06057848 was screened out as a hit compound by docking-based virtual screening and molecular dynamics (MD) simulation. The modification and optimization of hit ZINC06057848 resulted in the discovery of a series of novel 1,3,4-triazole-containing NA inhibitors (5a-5j). Compound 5c exerts the best inhibitory activity (IC50 = 0.11 µM) against NA, which is comparable to the positive control oseltamivir carboxylate (OSC) (IC50 = 0.10 µM). Molecular docking analysis indicates that the good efficacy of inhibitor 5c may be attributed to the furan and triazole rings extending into 430-cavity and the ethylbenzene part occupying the active site. The results of this work may help in the development of new NA inhibitors.


Assuntos
Acetamidas/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Triazóis/farmacologia , Acetamidas/síntese química , Acetamidas/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Neuraminidase/metabolismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química
12.
PLoS Negl Trop Dis ; 16(2): e0009585, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35130274

RESUMO

Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-ß-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-ß-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS.


Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Neuraminidase/química , Neuraminidase/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma congolense/enzimologia , Acetilgalactosamina/metabolismo , Sítios de Ligação , Catálise , Galactose/metabolismo , Glicoproteínas/genética , Neuraminidase/genética , Oligossacarídeos/metabolismo , Proteínas de Protozoários/genética , Ácidos Siálicos/metabolismo , Trypanosoma congolense/química , Trypanosoma congolense/genética
13.
Int J Mol Sci ; 23(3)2022 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-35162999

RESUMO

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3-/- in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.


Assuntos
Inibidores Enzimáticos/administração & dosagem , Vírus da Influenza A Subtipo H1N1/enzimologia , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Virais/metabolismo , Células A549 , Animais , Células Cultivadas , Modelos Animais de Doenças , Cães , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Células Madin Darby de Rim Canino , Camundongos , Neuraminidase/química , Infecções por Orthomyxoviridae/metabolismo , Cultura Primária de Células , Dobramento de Proteína , Traqueia/citologia , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Traqueia/virologia , Proteínas Virais/química
14.
Sci Rep ; 12(1): 505, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017592

RESUMO

Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. Here we report a virus enzyme assay as an alternative to these methods for detection of acute viral infection. In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. The resulting commercial test, the qFLU Dx Test, uses a different supply chain that does not compete with those for the current tests. The assay reagents were formulated as a master mix that accommodated both the neuraminidase and luciferase reactions, thereby enabling rapid and prolonged production of stable light signal in the presence of influenza virus in the sample. The assay was evaluated using depository throat swab specimens. As expected, the assay exhibited similar detection rates for all influenza types and subtypes except for A(H7N9), which exhibited lower detection rate due to lower viral titer in the specimens. When throat swab specimens were diluted with the sample buffer of the test kit and tested with the qFLU Dx Test. The sensitivity and specificity were 82.41% (95% confidence interval: 79.66-85.84%) and 95.39% (95% confidence interval: 94.32-96.46%), respectively, for these diluted specimens in comparison to a real-time polymerase chain reaction assay. The uniqueness of the qFLU Dx Test as an enzymatic assay makes it highly complementary with currently available methods.


Assuntos
Testes Diagnósticos de Rotina/métodos , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Influenza Humana/diagnóstico , Neuraminidase/análise , Proteínas Virais/análise , Testes Diagnósticos de Rotina/instrumentação , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Influenza Humana/virologia , Neuraminidase/genética , Neuraminidase/metabolismo , Faringe/virologia , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
J Virol ; 96(6): e0198221, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35045267

RESUMO

Many oseltamivir resistance mutations exhibit fitness defects in the absence of drug pressure that hinders their propagation in hosts. Secondary permissive mutations can rescue fitness defects and facilitate the segregation of resistance mutations in viral populations. Previous studies have identified a panel of permissive or compensatory mutations in neuraminidase (NA) that restore the growth defect of the predominant oseltamivir resistance mutation (H275Y) in H1N1 influenza A virus. In prior work, we identified a hyperactive mutation (Y276F) that increased NA activity by approximately 70%. While Y276F had not been previously identified as a permissive mutation, we hypothesized that Y276F may counteract the defects caused by H275Y by buffering its reduced NA expression and enzyme activity. In this study, we measured the relative fitness, NA activity, and surface expression, as well as sensitivity to oseltamivir, for several oseltamivir resistance mutations, including H275Y in the wild-type and Y276F genetic background. Our results demonstrate that Y276F selectively rescues the fitness defect of H275Y by restoring its NA surface expression and enzymatic activity, elucidating the local compensatory structural impacts of Y276F on the adjacent H275Y. IMPORTANCE The potential for influenza A virus (IAV) to cause pandemics makes understanding evolutionary mechanisms that impact drug resistance critical for developing surveillance and treatment strategies. Oseltamivir is the most widely used therapeutic strategy to treat IAV infections, but mutations in IAV can lead to drug resistance. The main oseltamivir resistance mutation, H275Y, occurs in the neuraminidase (NA) protein of IAV and reduces drug binding as well as NA function. Here, we identified a new helper mutation, Y276F, that can rescue the functional defects of H275Y and contribute to the evolution of drug resistance in IAV.


Assuntos
Farmacorresistência Viral , Vírus da Influenza A Subtipo H1N1 , Oseltamivir , Proteínas Virais , Antivirais/farmacologia , Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/genética , Influenza Humana/tratamento farmacológico , Mutação , Neuraminidase/genética , Neuraminidase/metabolismo , Oseltamivir/farmacologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
16.
Infect Immun ; 90(1): e0051621, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34662214

RESUMO

Salmonella invades and disrupts gut epithelium integrity, creating an infection-generated electric field that can drive directional migration of macrophages, a process called galvanotaxis. Phagocytosis of bacteria reverses the direction of macrophage galvanotaxis, implicating a bioelectrical mechanism to initiate life-threatening disseminations. The force that drives direction reversal of macrophage galvanotaxis is not understood. One hypothesis is that Salmonella can alter the electrical properties of the macrophages by modifying host cell surface glycan composition, which is supported by the fact that cleavage of surface-exposed sialic acids with a bacterial neuraminidase severely impairs macrophage galvanotaxis, as well as phagocytosis. Here, we utilize N-glycan profiling by nanoLC-chip QTOF mass cytometry to characterize the bacterial neuraminidase-associated compositional shift of the macrophage glycocalyx, which revealed a decrease in sialylated and an increase in fucosylated and high mannose structures. The Salmonella nanH gene, encoding a putative neuraminidase, is required for invasion and internalization in a human colonic epithelial cell infection model. To determine whether NanH is required for the Salmonella infection-dependent direction reversal, we constructed and characterized a nanH deletion mutant and found that NanH is partially required for Salmonella infection in primary murine macrophages. However, compared to wild type Salmonella, infection with the nanH mutant only marginally reduced the cathode-oriented macrophage galvonotaxis, without canceling direction reversal. Together, these findings strongly suggest that while neuraminidase-mediated N-glycan modification impaired both macrophage phagocytosis and galvanotaxis, yet to be defined mechanisms other than NanH may play a more important role in bioelectrical control of macrophage trafficking, which potentially triggers dissemination.


Assuntos
Quimiotaxia de Leucócito/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Neuraminidase/metabolismo , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Interações Hospedeiro-Patógeno/imunologia , Masculino , Camundongos , Modelos Biológicos , Mutação , Fagocitose/imunologia , Polissacarídeos/metabolismo , Infecções por Salmonella/microbiologia , Ácidos Siálicos/metabolismo , Virulência
17.
Comput Biol Med ; 141: 105149, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34953359

RESUMO

Seasonal human influenza is a serious respiratory infection caused by influenza viruses that can be found all over the world. Type A influenza is a contagious viral infection that, if left untreated, can lead to life-threatening consequences. Fortunately, the plant kingdom has many potent medicines with broad-spectrum antiviral activity. Herein, six plant constituents, namely Tanshinone IIA 1, Carnosic acid 2, Rosmarinic acid 3, Glycyrrhetinic acid 4, Baicalein 5, and Salvianolic acid B 6, were screened for their antiviral activities against H1N1 virus using in vitro and in silico approaches. Hence, their anti-influenza activities were tested in vitro to determine inhibitory concentration 50 (IC50) values after measuring their CC50 values using MTT assay on MDCK cells. Interestingly, Tanshinone IIA (TAN) 1 was the most promising member with CC50 = 9.678 µg/ml. Moreover, the plaque reduction assay carried on TAN 1 revealed promising viral inhibition percentages of 97.9%, 95.8%, 94.4%, and 91.7% using concentrations 0.05 µg/µl, 0.025 µg/µl, 0.0125 µg/µl, and 0.006 µg/µl, respectively. Furthermore, in silico molecular docking disclosed the superior affinities of Salvianolic acid B (SAL) 6 towards both surface glycoproteins of influenza A virus (namely, hemagglutinin (HA) and neuraminidase (NA)). The docked complexes of both SAL and TAN inside HA and NA receptor pockets were selected for 100 ns MD simulations followed by MM-GBSA binding free energy calculation to confirm the docking results and give more insights regarding the stability of both compounds inside influenza mentioned receptors, respectively. The selection criteria of the previously mentioned complexes were based on the fact that SAL showed the highest docking scores on both viral HA and NA glycoproteins whereas TAN achieved the best inhibitory activity on the other hand. Finally, we urge more advanced preclinical and clinical research, particularly for TAN, which could be used to treat the human influenza A virus effectively.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Abietanos , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A/metabolismo , Simulação de Acoplamento Molecular , Neuraminidase/metabolismo , Neuraminidase/farmacologia
18.
Lupus Sci Med ; 8(1)2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34872988

RESUMO

OBJECTIVE: Proteomic approach was applied to identify candidate biomarkers of chronicity in patients with proliferative lupus nephritis (LN), and their clinicopathological significance and prognostic values were investigated. METHODS: This study recruited 10 patients with proliferative LN and 6 normal controls (NCs) with proteomic data to compare protein expression profiles, 58 patients with proliferative LN and 10 NCs to verify proteomic data by immunohistochemistry, and 14 patients with proliferative LN with urine samples to evaluate the urinary expression of the biomarker by western blot assay. The composite endpoints included end-stage renal disease and ≥50% reduction from baseline estimated glomerular filtration rate (eGFR). RESULTS: Proteomics detected 48 proteins upregulated in the group with chronicity index (CI) ≥1 compared with the CI=0 and NC groups. Further pathway analysis was enriched in 'other glycan degradation'. Neuraminidase 1 (NEU1), the most predominant protein in the pathway of other glycan degradation, was highly expressed in the kidney of patients with proliferative LN and could co-localise with podocyte, mesangial cells, endothelial cells and tubule cells. NEU1 expression in the tubulointerstitium area was significantly higher in the CI ≥1 group compared with the CI=0 and NC groups. Moreover, NEU1 expression was significantly correlated with serum creatinine value, eGFR and CI scores, respectively. Urinary NEU1 excretion in the CI ≥1 group was higher than in the CI=0 group and was also positively correlated with CI scores. Furthermore, the high expression of renal NEU1 was identified as an independent risk factor for renal prognosis by multivariate Cox regression analysis (HR, 6.462 (95% CI 1.025 to 40.732), p=0.047). CONCLUSIONS: Renal NEU1 expression was associated with pathological CI scores and renal outcomes in patients with proliferative LN.


Assuntos
Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Biomarcadores , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Rim/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/diagnóstico , Neuraminidase/metabolismo , Proteômica
19.
Int J Mol Sci ; 22(23)2021 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-34884917

RESUMO

The growing resistance of the influenza virus to widely used competitive neuraminidase inhibitors occupying the active site of the enzyme requires the development of bifunctional compounds that can simultaneously interact with other regulatory sites on the protein surface. When developing such an inhibitor and combining structural fragments that could be located in the sialic acid cavity of the active site and the adjacent 430-cavity, it is necessary to select a suitable linker not only for connecting the fragments, but also to ensure effective interactions with the unique arginine triad Arg118-Arg292-Arg371 of neuraminidase. Using molecular modeling, we have demonstrated the usefulness of the sulfonamide group in the linker design and the potential advantage of this functional group over other isosteric analogues.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Neuraminidase/metabolismo , Orthomyxoviridae/enzimologia , Sulfonamidas/química , Antivirais/síntese química , Antivirais/química , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Orthomyxoviridae/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/metabolismo
20.
ACS Chem Biol ; 16(11): 2632-2640, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34724608

RESUMO

Sialidases catalyze the release of sialic acid from the terminus of glycan chains. We previously characterized the sialidase from the opportunistic fungal pathogen, Aspergillus fumigatus, and showed that it is a Kdnase. That is, this enzyme prefers 3-deoxy-d-glycero-d-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to N-acetylneuraminides (Neu5Ac). Here, we report characterization and crystal structures of putative sialidases from two other ascomycete fungal pathogens, Aspergillus terreus (AtS) and Trichophyton rubrum (TrS). Unlike A. fumigatus Kdnase (AfS), hydrolysis with the Neu5Ac substrates was negligible for TrS and AtS; thus, TrS and AtS are selective Kdnases. The second-order rate constant for hydrolysis of aryl Kdn glycosides by AtS is similar to that by AfS but 30-fold higher by TrS. The structures of these glycoside hydrolase family 33 (GH33) enzymes in complex with a range of ligands for both AtS and TrS show subtle changes in ring conformation that mimic the Michaelis complex, transition state, and covalent intermediate formed during catalysis. In addition, they can aid identification of important residues for distinguishing between Kdn and Neu5Ac substrates. When A. fumigatus, A. terreus, and T. rubrum were grown in chemically defined media, Kdn was detected in mycelial extracts, but Neu5Ac was only observed in A. terreus or T. rubrum extracts. The C8 monosaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) was also identified in A. fumigatus and T. rubrum samples. A fluorescent Kdn probe was synthesized and revealed the localization of AfS in vesicles at the cell surface.


Assuntos
Ascomicetos/enzimologia , Neuraminidase/metabolismo , Ascomicetos/crescimento & desenvolvimento , Catálise , Domínio Catalítico , Meios de Cultura , Estabilidade Enzimática , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Cinética , Neuraminidase/química , Conformação Proteica , Especificidade por Substrato , Temperatura
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