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Zinc plays a crucial role both in the immune system and endocrine processes. Zinc restriction in the diet has been shown to lead to degeneration of the endocrine pancreas, resulting in hormonal imbalance within the ß-cells. Proteostasismay vary depending on the stage of a pathophysiological process, which underscores the need for tools aimed at directly analyzing biological status. Among proteomics methods, MALDI-ToF-MS can serve as a rapid peptidomics tool for analyzing extracts or by histological imaging. Here we report the optimization of MALDI imaging mass spectrometry analysis of histological thin sections from mouse pancreas. This optimization enables the identification of the major islet peptide hormones as well as the major accumulated precursors and/or proteolytic products of peptide hormones. Cross-validation of the identified peptide hormones was performed by LC-ESI-MS from pancreatic islet extracts. Mice subjected to a zinc-restricted diet exhibited a relatively lower amount of peptide intermediates compared to the control group. These findings provide evidence for a complex modulation of proteostasis by micronutrients imbalance, a phenomenon directly accessed by MALDI-MSI.
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Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Zinco , Animais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Camundongos , Zinco/análise , Zinco/metabolismo , Hormônios Pancreáticos/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos Endogâmicos C57BL , Pâncreas/metabolismo , MasculinoRESUMO
The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1ß, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.
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Armadilhas Extracelulares , Infecção por Zika virus , Zika virus , Animais , Camundongos , Doenças Neuroinflamatórias , Nervo IsquiáticoRESUMO
OBJECTIVE: To detail the neurovascular crossing patterns between the leash of Henry (LoH) and the deep branch of the radial nerve (DBRN) in supination and pronation of the forearm, using imaging methods with anatomic correlation. MATERIALS AND METHODS: This cross-sectional study was performed ex vivo with HRUS and MRI with anatomic correlation on 6 samples and in vivo with HRUS with Doppler on 55 participants scanned bilaterally. The in vivo participants were enrolled over a 6-month period. The crossing patterns between the LoH and DBRN were assessed ex vivo and in vivo. Additional morphological features of the DBRN, LoH, and fat plane were assessed in vivo only. Biometric features of the participants were recorded. Statistical analyses were performed using Shapiro-Wilk, parametric and non-parametric tests. RESULTS: The most common neurovascular crossing pattern was the ascending branch of the radial recurrent artery (RRAab) crossing below (ex vivo: 83.3%, in vivo: 85.3%) and the muscular branch crossing above (ex vivo: 100%, in vivo: 63.2% %) the DBRN. Both the deep and superficial surfaces of the DBRN exhibited an intimate relationship with the vessels of the LoH. A positive correlation between vessel diameter and anthropometric factors was observed. In addition, the muscular branch exhibited a significantly smaller diameter than the RRAab. CONCLUSION: Our study detailed the relationship between the LoH and the DBRN and highlighted the high incidence of vessel crossing above the DBRN at the level of the muscular branch. Knowledge of neurovascular crossings is crucial for understanding neurovascular entrapment syndromes and planning interventional procedures to reduce vascular complications.
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RATIONALE: Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) of tissues became popular in the last decade. Consequently, adapting sample preparation methods for different materials turned out to be a pivotal step for successful analysis due to the requirement of sample slices of 12-20 µm thickness. However, acquiring thin sections compatible with MALDI-IMS for unusual samples is challenging, as existing histological protocols may not be suitable, thus requiring new methods. Açaí (Euterpe oleracea Mart.) seed is an example of a challenging material due to its toughness and resistance to crack, therefore our goal was to develop a methodology to obtain thin (12-20 µm) and entire sections of açaí seeds for MALDI-IMS analysis. METHODS: Different strategies were evaluated for obtaining thin sections of seeds, and the combination of the following steps was found to be the most suitable option: (i) softening of seeds by water immersion for 24 h; (ii) transversal cut of seeds to obtain half-seeds using a razor blade and a hammer; (iii) half-seeds imbibition in gelatin; (iv) samples sectioning using a cryostat at -20°C to obtain samples with 12-20 µm thickness; (v) collection of samples in an indium tin oxide-coated glass slide covered by double-sided copper tape to avoid sample wrapping and ensure adhesion after unfreezing; and (vi) storage of samples in a -80°C freezer, if necessary. RESULTS: This adapted sample preparation method enabled the analysis of açaí seeds by MALDI-IMS, providing spatial distribution of carbohydrates in the endosperm. CONCLUSIONS: The adaptations developed for sample preparation will help investigate the metabolic and physiological properties of açaí seeds in future studies.
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OBJECTIVE: To evaluate the effect of maximal pronation and supination of the forearm on the alignment and anatomic relationship of the deep branch of the radial nerve (DBRN) at the superior arcade of the supinator muscle (SASM) by using high-resolution ultrasound (HRUS). MATERIALS AND METHODS: In this cross-sectional study, HRUS in the long axis of the DBRN was performed in asymptomatic participants enrolled from March to August 2021. DBRN alignment was evaluated by measuring angles of the nerve in maximal pronation and maximal supination of the forearm independently by two musculoskeletal radiologists. Forearm range of motion and biometric measurements were recorded. Student t, Shapiro-Wilk, Pearson correlation, reliability analyses, and Kruskal-Wallis test were used. RESULTS: The study population included 110 nerves from 55 asymptomatic participants (median age, 37.0 years; age range, 16-63 years; 29 [52.7%] women). There was a statistically significant difference between the DBRN angle in maximal supination and maximal pronation (Reader 1: 95% CI: 5.74, 8.21, p < 0.001, and Reader 2: 95% CI: 5.82, 8.37, p < 0.001). The mean difference between the angles in maximal supination and maximal pronation was approximately 7° for both readers. ICC was very good for intraobserver agreement (Reader1: r ≥ 0.92, p < 0.001; Reader 2: r ≥ 0.93, p < 0.001), as well as for interobserver agreement (phase 1: r ≥ 0.87, p < 0.001; phase 2: r ≥ 0.90, p < 0.001). CONCLUSION: The extremes of the rotational movement of the forearm affect the longitudinal morphology and anatomic relationships of the DBRN, primarily demonstrating the convergence of the nerve towards the SASM in maximal pronation and divergence in maximal supination.
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Antebraço , Nervo Radial , Humanos , Feminino , Adulto , Adolescente , Adulto Jovem , Pessoa de Meia-Idade , Masculino , Nervo Radial/diagnóstico por imagem , Nervo Radial/anatomia & histologia , Pronação , Supinação , Estudos Transversais , Reprodutibilidade dos Testes , Cadáver , Antebraço/diagnóstico por imagem , Antebraço/inervaçãoRESUMO
BACKGROUND: Dengue is a disease caused by dengue virus (DENV-1 through -4). Among the four serotypes, DENV-4 remains the least studied. Acute kidney injury is a potential complication of dengue generally associated with severe dengue infection. OBJECTIVES: The goal of this study was to investigate the alterations caused by experimental dengue infection in the kidney of adult BALB/c mice. METHODS: In this study, BALB/c mice were infected through the intravenous route with a DENV-4 strain, isolated from a human patient. The kidneys of the mice were procured and subject to histopathological and ultrastructural analysis. FINDINGS: The presence of the viral antigen was confirmed through immunohistochemistry. Analysis of tissue sections revealed the presence of inflammatory cell infiltrate throughout the parenchyma. Glomerular enlargement was a common find. Necrosis of tubular cells and haemorrhage were also observed. Analysis of the kidney on a transmission electron microscope allowed a closer look into the necrotic tubular cells, which presented nuclei with condensed chromatin, and loss of cytoplasm. MAIN CONCLUSIONS: Even though the kidney is probably not a primary target of dengue infection in mice, the inoculation of the virus in the blood appears to damage the renal tissue through local inflammation.
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Vírus da Dengue , Dengue Grave , Adulto , Humanos , Animais , Camundongos , Rim , Antígenos Virais , Camundongos Endogâmicos BALB CRESUMO
Glycoconjugates play a central role in several cellular processes, and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutamine:fructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in Escherichia coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics, and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.
Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos , Multimerização ProteicaRESUMO
Dengue is a mosquito-borne infectious disease that is highly endemic in tropical and subtropical countries. Symptomatic patients can rapidly progress to severe conditions of hemorrhage, plasma extravasation, and hypovolemic shock, which leads to death. The blood tests of patients with severe dengue typically reveal low levels of high-density lipoprotein (HDL), which is responsible for reverse cholesterol transport (RCT) and regulation of the lipid composition in peripheral tissues. It is well known that dengue virus (DENV) depends on membrane cholesterol rafts to infect and to replicate in mammalian cells. Here, we describe the interaction of DENV nonstructural protein 1 (NS1) with apolipoprotein A1 (ApoA1), which is the major protein component of HDL. NS1 is secreted by infected cells and can be found circulating in the serum of patients with the onset of symptoms. NS1 concentrations in plasma are related to dengue severity, which is attributed to immune evasion and an acute inflammatory response. Our data show that the DENV NS1 protein induces an increase of lipid rafts in noninfected cell membranes and enhances further DENV infection. We also show that ApoA1-mediated lipid raft depletion inhibits DENV attachment to the cell surface. In addition, ApoA1 is able to neutralize NS1-induced cell activation and to prevent NS1-mediated enhancement of DENV infection. Furthermore, we demonstrate that the ApoA1 mimetic peptide 4F is also capable of mediating lipid raft depletion to control DENV infection. Taken together, our results suggest the potential of RCT-based therapies for dengue treatment. These results should motivate studies to assess the importance of RCT in DENV infection in vivo. IMPORTANCE DENV is one of the most relevant mosquito-transmitted viruses worldwide, infecting more than 390 million people every year and leading to more than 20 thousand deaths. Although a DENV vaccine has already been approved, its potential side effects have hampered its use in large-scale immunizations. Therefore, new treatment options are urgently needed to prevent disease worsening or to improve current clinical management of severe cases. In this study, we describe a new interaction of the NS1 protein, one of the major viral components, with a key component of HDL, ApoA1. This interaction seems to alter membrane susceptibility to virus infection and modulates the mechanisms triggered by DENV to evade the immune response. We also propose the use of a mimetic peptide named 4F, which was originally developed for atherosclerosis, as a potential therapy for relieving DENV symptoms.
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Apolipoproteína A-I/imunologia , Vírus da Dengue/metabolismo , Evasão da Resposta Imune/imunologia , Microdomínios da Membrana/metabolismo , Proteínas não Estruturais Virais/imunologia , Animais , Antivirais/farmacologia , Linhagem Celular , Colesterol/metabolismo , Dengue/patologia , Humanos , Inflamação/prevenção & controle , Camundongos , Peptídeos/farmacologia , Células RAW 264.7 , Ligação Viral/efeitos dos fármacosRESUMO
Gangliosides are glycosphingolipids abundantly expressed in the vertebrate nervous system, and are classified into a-, b-, or c-series according to the number of sialic acid residues. The enzyme GD3 synthase converts GM3 (an a-series ganglioside) into GD3, a b-series ganglioside highly expressed in the developing and adult retina. The present study evaluated the visual system of GD3 synthase knockout mice (GD3s-/- ), morphologically and functionally. The absence of b- series gangliosides in the retinas of knockout animals was confirmed by mass spectrometry imaging, which also indicated an accumulation of a-series gangliosides, such as GM3. Retinal ganglion cell (RGC) density was significantly reduced in GD3s-/- mice, with a similar reduction in the number of axons in the optic nerve. Knockout animals also showed a 15% reduction in the number of photoreceptor nuclei, but no difference in the bipolar cells. The area occupied by GFAP-positive glial cells was smaller in GD3s-/- retinas, but the number of microglial cells/macrophages did not change. In addition to the morphological alterations, a 30% reduction in light responsiveness was detected through quantification of pS6-expressing RGC, an indicator of neural activity. Furthermore, electroretinography (ERG) indicated a significant reduction in RGC and photoreceptor electrical activity in GD3s-/- mice, as indicated by scotopic ERG and pattern ERG (PERG) amplitudes. Finally, evaluation of the optomotor response demonstrated that GD3s-/- mice have reduced visual acuity and contrast sensitivity. These results suggest that b-series gangliosides play a critical role in regulating the structure and function of the mouse visual system.
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Sensibilidades de Contraste/fisiologia , Deleção de Genes , Retina/enzimologia , Sialiltransferases/deficiência , Sialiltransferases/genética , Acuidade Visual/fisiologia , Animais , Eletrorretinografia/métodos , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Estimulação Luminosa/métodosRESUMO
Interferon response suppression by the respiratory syncytial virus relies on two unique nonstructural proteins, NS1 and NS2, that interact with cellular partners through high-order complexes. We hypothesized that two conserved proline residues, P81 and P67, participate in the conformational change leading to oligomerization. We found that the molecular dynamics of NS1 show a highly mobile C-terminal helix, which becomes rigid upon in silico replacement of P81. A soluble oligomerization pathway into regular spherical structures at low ionic strengths competes with an aggregation pathway at high ionic strengths with an increase in temperature. P81A requires higher temperatures to oligomerize and has a small positive effect on aggregation, while P67A is largely prone to aggregation. Chemical denaturation shows a first transition, involving a high fluorescence and ellipticity change corresponding to both a conformational change and substantial effects on the environment of its single tryptophan, that is strongly destabilized by P67A but stabilized by P81A. The subsequent global cooperative unfolding corresponding to the main ß-sheet core is not affected by the proline mutations. Thus, a clear link exists between the effect of P81 and P67 on the stability of the first transition and oligomerization/aggregation. Interestingly, both P67 and P81 are located far away in space and sequence from the C-terminal helix, indicating a marked global structural dynamics. This provides a mechanism for modulating the oligomerization of NS1 by unfolding of a weak helix that exposes hydrophobic surfaces, linked to the participation of NS1 in multiprotein complexes.
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Interferons/imunologia , Prolina/química , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/química , Proteínas não Estruturais Virais/química , Humanos , Isomerismo , Modelos Moleculares , Prolina/imunologia , Conformação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Desdobramento de Proteína , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas não Estruturais Virais/imunologiaRESUMO
The dengue virus (DENV) non structural protein 1 (NS1) is a 46-55â¯kDa protein that exists as homodimer inside cells and as hexamer in the extracellular milieu. Several lines of evidence have demonstrated that the biochemical and structural properties of recombinant NS1 (rNS1) vary depending on the protein expression system used. Aiming to improve current tools for studying NS1 multiple roles, a recombinant tag-free NS1 protein was expressed and purified from P. pastoris yeast cells. The best expression condition was achieved using GS115 strain and induction for 72â¯h with 0.7% methanol addition every 24â¯h. Total secreted rNS1 reached 2.18â¯mg in 1â¯L culture and the final yield of the purified protein was 1â¯mg per liter of culture (recovery yield of approximately 45.9%). Size-exclusion chromatography and treatment with EndoH and PNGase indicate that it exists as an N-glycosylated homodimer. Moreover, an ELISA assay with specific DENV anti-NS1 antibody that recognizes conformational epitopes revealed that rNS1 has most of its conformational epitopes preserved. The expression of rNS1 in its native conformation is an important tool for further studies of its role in DENV pathogenesis and replication.
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Vírus da Dengue/metabolismo , Pichia/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Cromatografia em Gel , Dengue/virologia , Vírus da Dengue/química , Vírus da Dengue/genética , Expressão Gênica , Glicosilação , Humanos , Pichia/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismoRESUMO
Yellow fever virus (YFV) replication is highly dependent on host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1-DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein that has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner. 59 proteins were found to be significantly increased when compared with a negative control, and CypA exhibited the greatest difference, with a 22-fold change. Fisher's exact test was significant for 58 proteins, and the p value of CypA was the most significant (0.000000019). The Ingenuity Systems software identified 16 pathways, and this analysis indicated sirolimus, an mTOR pathway inhibitor, as a potential inhibitor of CypA. Immunofluorescence and viral plaque assays showed a significant reduction in YFV replication using sirolimus and cyclosporine A (CsA) as inhibitors. Furthermore, YFV replication was strongly inhibited in cells treated with both inhibitors using reporter BHK-21-rep-YFV17D-LucNeoIres cells. Taken together, these data suggest that CypA-NS4B interaction regulates YFV replication. Finally, we present the first evidence that YFV inhibition may depend on NS4B-CypA interaction.
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Ciclofilina A/metabolismo , Proteínas/genética , Replicação Viral/genética , Vírus da Febre Amarela/genética , Ciclofilina A/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Biologia de Sistemas , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Vírus da Febre Amarela/patogenicidadeRESUMO
Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE: Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system.
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Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Vírus da Dengue/metabolismo , Dengue/metabolismo , Dengue/virologia , Vitronectina/metabolismo , Linhagem Celular Tumoral , Flavivirus/metabolismo , Humanos , Ligação Proteica/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/metabolismo , Zika virus/metabolismo , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologiaRESUMO
Hepatitis C infection is a cause of chronic liver diseases such as cirrhosis and carcinoma. The current therapy for hepatitis C has limited efficacy and low tolerance. The HCV encodes a serine protease which is critical for viral replication, and few protease inhibitors are currently on the market. In this paper, we describe the synthesis and screening of novel isosorbide-based peptidomimetic inhibitors, in which the compounds 1d, 1e, and 1i showed significant inhibition of the protease activity in vitro at 100µM. The compound 1e also showed dose-response (IC50=36±3µM) and inhibited the protease mutants D168A and V170A at 100µM, indicating it as a promising inhibitor of the HCV NS3/4A protease. Our molecular modeling studies suggest that the activity of 1e is associated with a change in the interactions of S2 and S4 subsites, since that the increased flexibility favors a decrease in activity against D168A, whereas the appearance of a hydrophobic cavity in the S4 subsite increase the inhibition against V170A strain.
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Antivirais/química , Hepacivirus/enzimologia , Isossorbida/química , Serina Proteases/química , Inibidores de Serina Proteinase/química , Antivirais/síntese química , Antivirais/farmacologia , Sítios de Ligação , Domínio Catalítico , Hepacivirus/efeitos dos fármacos , Isossorbida/síntese química , Isossorbida/farmacologia , Simulação de Acoplamento Molecular , Mutação , Peptidomiméticos , Serina Proteases/genética , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The present report describes a case of splenic rupture due to dengue, a rare complication of dengue that should be considered in any patient with suspected dengue disease who started with left upper quadrant abdominal pain and hypotension. The pathophysiology of this entity is not yet well elucidated, but one of the theories present in the literature is that it is due to a depletion of coagulation factors and platelets leading to intra-splenic hemorrhage and rupture. The RT-PCR technique detected serotype 1 and histopathological studies of the spleen revealed significant atrophy of lymphoid follicles and extensive hemorrhage areas. Besides histopathological observations, virus replication was investigated by detection of dengue antigens, especially the non-structural 3 protein (NS3) in endothelial cells and splenic macrophages. This important complication has serious clinical repercussions and high mortality, due to the diagnostic difficulty and many factors that usually confuse or delay its diagnosis. Therefore, it is of the utmost importance to recognize their manifestations and their management to try to best minimize their consequences and mortality.
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Dengue/complicações , Ruptura Esplênica/etiologia , Replicação Viral/fisiologia , Humanos , Masculino , Ruptura Esplênica/patologia , Ruptura Esplênica/cirurgia , Adulto JovemRESUMO
During infection, human immunodeficiency virus type 1 (HIV-1) interacts with the cellular host factor cyclophilin A (CypA) through residues 85-93 of the N-terminal domain of HIV-1's capsid protein (CA). The role of the CA:CypA interaction is still unclear. Previous studies showed that a CypA-binding loop mutant, Δ87-97, has increased ability to assemble in vitro. We used this mutant to infer whether the CypA-binding region has an overall effect on CA stability, as measured by pressure and chemical perturbation. We built a SAXS-based envelope model for the dimer of both WT and Δ87-97. A new conformational arrangement of the dimers is described, showing the structural plasticity that CA can adopt. In protein folding studies, the deletion of the loop drastically reduces CA stability, as assayed by high hydrostatic pressure and urea. We hypothesize that the deletion promotes a rearrangement of helix 4, which may enhance the heterotypic interaction between the N- and C-terminal domains of CA dimers. In addition, we propose that the cyclophilin-binding loop may modulate capsid assembly during infection, either in the cytoplasm or near the nucleus by binding to the nuclear protein Nup385.
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Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Ciclofilina A/metabolismo , HIV-1/química , HIV-1/fisiologia , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Espalhamento a Baixo Ângulo , Montagem de Vírus , Difração de Raios XRESUMO
UNLABELLED: Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE: Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and the extracellular milieus. Despite the fact that NS1 has been commonly associated with DENV pathogenesis, it plays a pivotal but unknown role in the replication process. In an effort to understand the role of intracellular NS1, we demonstrate that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with NS1. Our results indicate that NS1 increases the glycolytic activity of GAPDH in vitro. Interestingly, the GAPDH activity was increased during DENV infection, and NS1 expression alone was sufficient to enhance intracellular GAPDH activity in BHK-21 cells. Overall, our findings suggest that NS1 is an important modulator of cellular energy metabolism by increasing glycolytic flux.
Assuntos
Metabolismo Energético/fisiologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Modelos Moleculares , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Análise de Variância , Animais , Linhagem Celular , Cromatografia Líquida , Cricetinae , Ensaio de Imunoadsorção Enzimática , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Ligação Proteica , Espectrometria de Massas em Tandem , Proteínas não Estruturais Virais/genéticaRESUMO
The NS3 protein is a multifunctional non-structural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a His-tagged fusion protein in Escherichia coli. The pNS3 protein was purified by two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-α in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques. The successfully purified recombinant protein was able to preserv the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.
Assuntos
Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Vacinas Sintéticas/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Western Blotting , Clonagem Molecular , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/genética , Vacinas contra Dengue/isolamento & purificação , Vírus da Dengue/genética , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Feminino , Imunofluorescência , Expressão Gênica , Interferon gama/metabolismo , Interleucina-10/metabolismo , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , RNA Helicases/genética , RNA Helicases/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/isolamento & purificação , Proteínas não Estruturais Virais/genéticaRESUMO
Dengue affects millions of people worldwide. No specific treatment is currently available, in part due to an incomplete understanding of the viral components' interactions with host cellular structures. We tested dengue virus (DENV) capsid protein (C) interaction with low- and very low-density lipoproteins (LDL and VLDL, respectively) using atomic force microscopy-based force spectroscopy, dynamic light scattering, NMR and computational analysis. Data reveal a specific DENV C interaction with VLDL, but not LDL. This binding is potassium-dependent and involves the DENV C N-terminal region, as previously observed for the DENV C-lipid droplets (LDs) interaction. A successful inhibition of DENV C-VLDL binding was achieved with a peptide drug lead. The similarities between LDs and VLDL, and between perilipin 3 (DENV C target on LDs) and ApoE, indicate ApoE as the molecular target on VLDL. We hypothesize that DENV may form lipoviroparticles, which would constitute a novel step on DENV life cycle. FROM THE CLINICAL EDITOR: Using atomic force microscopy-based force spectroscopy, dynamic light scattering, NMR, and computational analysis, these authors demonstrate that dengue viral capsid proteins (DENV C) bind to very low density lipoprotein surfaces, but not to LDLs, in a potassium-dependent manner. This observation suggests the formation of lipo-viroparticles, which may be a novel step in its life cycle, and may offer potential therapeutic interventions directed to this step.
Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Dengue/metabolismo , Dengue/virologia , Lipoproteínas VLDL/metabolismo , Dengue/genética , Dengue/patologia , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Potássio/metabolismo , Ligação Proteica , Vírion/genética , Vírion/metabolismoRESUMO
The Zika virus (ZIKV) is a global health threat due to its rapid spread and severe health implications, including congenital abnormalities and neurological complications. Differentiating ZIKV from other arboviruses such as dengue virus (DENV) is crucial for effective diagnosis and treatment. This study presents the development of a biosensor for detecting the ZIKV non-structural protein 1 (NS1) using gold nanoparticles (AuNPs) functionalized with monoclonal antibodies employing dynamic light scattering (DLS). The biosensor named ZINS1-mAb-AuNP exhibited specific binding to the ZIKV NS1 protein, demonstrating high colloidal stability indicated by a hydrodynamic diameter (DH) of 140 nm, detectable via DLS. In the absence of the protein, the high ionic strength medium caused particle aggregation. This detection method showed good sensitivity and specificity, with a limit of detection (LOD) of 0.96 µg mL-1, and avoided cross-reactivity with DENV2 NS1 and SARS-CoV-2 spike proteins. The ZINS1-mAb-AuNP biosensor represents a promising tool for the early and accurate detection of ZIKV, facilitating diagnostic and treatment capabilities for arboviral infections.