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Natural Health Products (NHPs) have long been considered a valuable therapeutic approach for the prevention and treatment of various diseases, including cancer. However, research on this topic has led to inconclusive and often controversial results. This review aims to provide a comprehensive update of the effects and mechanisms related to the use of NHPs, to describe the results of randomized clinical trials (RCTs) on their effects in cancer patients, and to critically discuss factors influencing clinical outcomes. RCTs available in the literature, even those studying the same NHP, are very heterogeneous in terms of indications, doses, route and timing of administration, and outcomes evaluated. Silymarin, ginsenoside, and vitamin E appear to be useful in attenuating adverse events related to radiotherapy or chemotherapy, and curcumin and lycopene might provide some benefit in patients with prostate cancer. Most RCTs have not clarified whether NHP supplementation provides any real benefit, while harmful effects have been shown in some cases. Overall, the available data suggest that although there is some evidence to support the benefits of NHPs in the management of cancer patients, further clinical trials with the same design are needed before their introduction into clinical practice can be considered.
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The identification of archaeological biomarkers is one of the main objectives of analytical chemistry in the archaeological field. However, no information is currently available on biomarkers able to unambiguously indicate the presence of olive oil, a cornerstone of Mediterranean ancient societies lifestyle, in an organic residue. This study aims to bridge this gap by a thorough characterization of the degradation products of extra-virgin olive oils (EVOOs) resulting from in-lab thermal oxidative treatments, with the primary goal of revealing potential archaeological biomarkers for olive oil. Thirty-three EVOOs sourced from eleven different monocultivars across five Italian regions (Sicily, Apulia, Lazio, Tuscany, and Liguria) and Spain, were analyzed before and after thermal oxidation. In addition, an identical thermal treatment was employed on pure triglyceride standards (triolein, trilinolein, and tristearin), due to the high concentration of their fatty acids in EVOO discerning their degradation patterns. A combination of analytical strategies was employed, including HPLC-MS and HPLC-ELSD for the complete evaluation of the intact lipids (triglycerides, diglycerides, and their oxidative species) in olive oils before and after oxidation, and HS-SPME-GC-MS and GC-FID for the characterization of secondary oxidation products formed by the thermal treatment. In addition, to elucidate the fatty acid distribution in the oxidized EVOOs by GC-MS and GC-FID techniques a derivatization step was performed to convert lipid compounds into trimethylsilyl (TMS) derivatives. A chemometric approach was used to thoroughly interpret the data obtained from intact and oxidized samples. This comprehensive investigation sheds light on the chemical transformations of EVOOs under thermal oxidative conditions and indicates mono-carboxylic acids such as pentanoic, hexanoic, heptanoic, octanoic, nonanoic, and decanoic acids as potential archaeological biomarkers for the presence of lipid substances coming from olive oil in archaeological organic residues. Finally, lipid contents from twenty-four real archaeological samples, grouped in amphorae (10), unguentaria (5), and lamps (9), excavated from the Roman domus of Villa San Pancrazio in Taormina (Italy), were determined. The analytical results obtained from amphorae samples revealed the presence of the selected olive oil-specific archaeological biomarkers, an information extremely interesting considering that this type of amphorae have so far been solely associated with the storage of wine.
Assuntos
Arqueologia , Azeite de Oliva , Oxirredução , Azeite de Oliva/química , Itália , Arqueologia/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Lipídeos/química , Lipídeos/análise , Ácidos Graxos/análise , Ácidos Graxos/química , Espectrometria de Massas/métodosRESUMO
The validity of omega 3 fatty acids (ω3 FAs), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as dietary supplements has been widely proved. It's well known in fact, that they protect against cardiovascular diseases, reduce the levels of triacylglycerides (TAGs) and cholesteryl esters (CEs) in blood, and have anti-inflammatory activity. For these reasons, in the last few years the production of dietary supplement containing ω3 has increased significantly. In this context, the possibility to obtain ω3 and other high value molecules from alternative sources as fish waste, in accordance with the principles of circular economy, becomes an enormous attractive. In addition, the opportunity of creating new products, with greater health benefits, represents an interesting challenge. The current study was focused on the extraction of ω3 fatty acids and peptides from tuna waste industry, to realize a new dietary supplement. To this purpose, a supercritical fluid extraction (SFE) method was developed to separate, isolate, and enrich the different fractions subsequently used to produce an innovative formulate. The obtained supplement was characterized in terms of fatty acids esterified ester (FAEE) composition by gas chromatography (GC) coupled to both flame ionization detection (FID) and mass spectrometry (MS), and content of heavy metals by inductively coupled plasma-mass spectrometry (ICP-MS). The effects of ω3 supplementation on metabolism and circulating lipid profiles was tested on 12 volunteers and assessed by GC-FID analysis of whole blood collected on paper support (Dried Blood Spot, DBS) at the beginning of the study and after thirty days. The results of plasma fatty acids levels after 30 days showed a significant decrease in the ω6/ω3 ratio, as well as the saturated/polyunsaturated fatty acids (SFA/PUFA) ratio, compared to subjects who took the ω3 ethyl esters unformulated. The novel formulated supplements proved to be extremely interesting and promising products, due to a significant increase in bioavailability, that makes it highly competitive in the current panorama of the nutraceutical industry.
Assuntos
Ésteres , Ácidos Graxos Ômega-3 , Animais , Humanos , Cromatografia Gasosa-Espectrometria de Massas , Ácido Eicosapentaenoico , Ácidos Graxos , Suplementos NutricionaisRESUMO
The balance between the different lipid molecules present in biological fluids accurately reflects the health state of the organism and can be used by medical personnel to finely tune therapy to a single patient, a process known as precision medicine. In this work, we developed a miniaturized workflow for the analysis of different lipid classes at the intact level, as well as their fatty acid constituents, starting from human serum. Fatty acids were identified by using flow-modulated comprehensive gas chromatography coupled to mass spectrometry (FM-GC × GC-MS), and their relative amount as well as the ratio of specific FA classes was determined by using FM-GC × GC with a flame ionization detector. Ultra-high-performance liquid chromatography coupled to tandem mass spectrometry was used for the simultaneous quantification of vitamin D metabolites and assessment of different intact lipid classes. An MRM method was developed for the quantification of five vitamin D metabolites (vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3), and validated in terms of LoD, LoQ, accuracy, and precision, also using a certified reference material. At the same time, a combination of SCAN, precursor ion scan, and neutral loss scan, in both positive and negative modes, was used for the identification of 81 intact lipid species, such as phospholipids, cholesteryl esters, and triacylglycerols, in less than 25 min. In order to easily monitor the lipid composition and speed up the identification process, a two-dimensional map of the lipidome was generated, by plotting the molecular weight of the identified molecules versus their retention time. Moreover, a relative quantification was performed within each lipid class identified. The combination of untargeted and targeted data could provide useful information about the pathophysiological condition of the organism and evaluate, in a tailored manner, an efficient action.
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Lipidômica , Vitamina D , Humanos , Vitaminas , Calcifediol , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Moraxella catarrhalis is an important and common respiratory pathogen that can cause Otitis Media, Community Acquired Pneumonia, and has been associated with an increased risk of exacerbations in chronic obstructive pulmonary disease in adults, leading to morbidity and mortality. Its ubiquitous surface protein A2 (UspA2) has been shown to interact with host structures and extracellular matrix proteins, suggesting a role at an early stage of infection and a contribution to bacterial serum resistance. The UspA proteins are homo-trimeric autotransporters that appear as a lollipop-shaped structure in electron micrographs. They are composed of an N-terminal head with adhesive properties, followed by a stalk, which ends by an amphipathic helix and a C-terminal membrane domain. The three family members UspA1, UspA2 and UspA2H, present different amino acid signatures both at the head and membrane-spanning regions. By combining electron microscopy, hydrogen deuterium exchange mass spectrometry and protein modeling, we identified a shared and repeated epitope recognized by FHUSPA2/10, a potent cross-bactericidal monoclonal antibody raised by UspA2 and deduced key amino acids involved in the binding. The finding strengthens the potential of UspA2 to be incorporated in a vaccine formulation against M. catarrhalis.
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Antibacterianos , Anticorpos Monoclonais , Moraxella catarrhalis , Adulto , Humanos , Aminoácidos/metabolismo , Anticorpos Monoclonais/farmacologia , Proteínas da Membrana Bacteriana Externa/imunologia , Epitopos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Sistemas de Secreção Tipo V/metabolismo , Antibacterianos/farmacologiaRESUMO
With the onset of Listeria monocytogenes resistance to the bacteriocin nisin, the search for alternative antimicrobial treatments is of fundamental importance. In this work, we set out to investigate proteins and lipids involved in the resistance mechanisms of L. monocytogenes against the antimicrobial peptides (AMPs) nisin and fengycin. The effect of sub-lethal concentrations of nisin and lipopeptide fengycin secreted by Bacillus velezensis P34 on L. monocytogenes was investigated by mass spectrometry-based lipidomics and proteomics. Both AMPs caused a differential regulation of biofilm formation, confirming the promotion of cell attachment and biofilm assembling after treatment with nisin, whereas growth inhibition was observed after fengycin treatment. Anteiso branched-chain fatty acids were detected in higher amounts in fengycin-treated samples (46.6%) as compared to nisin-treated and control samples (39.4% and 43.4%, respectively). In addition, a higher relative abundance of 30:0, 31:0 and 32:0 phosphatidylglycerol species was detected in fengycin-treated samples. The lipidomics data suggest the inhibition of biofilm formation by the fengycin treatment, while the proteomics data revealed downregulation of important cell wall proteins involved in the building of biofilms, such as the lipoteichoic acid backbone synthesis (Lmo0927) and the flagella-related (Lmo0718) proteins among others. Together, these results provide new insights into the modification of lipid and protein profiles and biofilm formation in L. monocytogenes upon exposure to antimicrobial peptides.
Assuntos
Bacteriocinas , Listeria monocytogenes , Nisina , Peptídeos Antimicrobianos , Lipídeos , Listeria monocytogenes/fisiologia , Nisina/farmacologiaRESUMO
The use of IRMS as a GC detector has a history going back decades, however the critical issue of wrong δ13C measurements resulting from impure peaks has been often underestimated. To this regard, multidimensional separation techniques are effective tools to improve the reliability of the data, with respect to those obtained after monodimensional analysis. The present research aims to draw attention to one critical issue, related to the reliability of the δ13C data obtained by means of monodimensional GC-C-IRMS. Although already known from the literature, such aspect has been greatly overlooked, as is reflected in the few papers reporting the use of MDGC, among the plethora of published research dealing with GC-C-IRMS applications. Hereby, a set of natural samples of complex composition were analysed to investigate the presence of minor or even undetected coelutions, and to which extent it affected the isotope ratio determination. Apart from chromatographic effects, and issues related to analytes conversion to CO2 prior to IRMS measurement, unpredictable co-elutions with compounds, either resulting from oxidation or intentionally added in fraudulent practices, could also contribute to a shift of the δ13C data, up to 10 and higher. Last, the influence of column bleed was investigated, as affecting the determination of the δ13C data for compounds that were eluted at high temperatures. It was finally demonstrated by the selected key studies that implementation of MDGC separation is mandatory to prevent the aforementioned issues, aiming to guarantee accurate results. In the light of the above conclusions, and considering the level of automation of heart-cut devices nowadays available, routine practice of MDGC results highly recommendable in any IRMS applications.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Isótopos de Carbono , Espectrometria de Massas , Reprodutibilidade dos TestesRESUMO
Comprehensive two-dimensional liquid chromatography is a well-established method for the unraveling of very complex real-world samples. With regard to food and natural products such a technique turned out to be a very promising approach due to its high resolving power and improved identification capability, especially in combination with mass spectrometry. In this context, polyphenols comprise a particular complex class of bioactive compounds, due to their nature and content in commonly consumed foodstuffs, making their analysis challenging. The present contribution shows an overview of the two commonly employed approaches used for polyphenol analysis, viz. RP-LC × RP-LC and HILIC × RP-LC. Furthermore, the latest implementations as well as limitations and future perspectives are critically reported.
Assuntos
Produtos Biológicos/química , Cromatografia Líquida/métodos , Análise de Alimentos/métodos , Polifenóis/análise , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de MassasRESUMO
The present work aims to a promising re-utilization of the massive waste derived from the tuna fishing industry, for which by-products can represent more than 50% of the original material. Due to the considerable content in polyunsaturated fatty acids and noble proteins, such wastes can be used as primary source of functional ingredients in the production of nutraceuticals. The composition of the lipid and protein tuna fractions was investigated by means of gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry methods (in wastes and edible parts), and a preliminary characterization of potential bioactive peptides was achieved. Automated sample preparation allowed speeding up the analytical workflow, while allowing for highly sensitive and selective lipid characterization. The ω3 fatty acid content was found higher in waste products compared to the muscle, in terms of fatty acids as well as complex lipids. As for peptides, extraction by isoelectric solubilization/precipitation was performed, followed by enzymatic digestion and high-performance liquid chromatography-tandem mass spectrometry analysis. Furthermore, the use of bioinformatics tools highlighted the presence of potential antimicrobial peptides in the samples investigated.
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Automação , Lipídeos/análise , Proteínas/análise , Resíduos/análise , Animais , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Pesqueiros , Indústrias , AtumRESUMO
The global Cannabis Sativa market, including essential oils, foods, personal-care products, and medical formulations has gained much attention over the last years due to the favorable regulatory framework. Undoubtedly, the enormous interest about cannabis cultivation mainly derives from the well-known pharmacological properties of cannabinoids and terpenes biosynthesized by the plants. In this review, the most recently used analytical methodologies for detecting both cannabinoids and terpenes are described. Well-established and innovative extraction protocols, and chromatographic separations, such as GC and HPLC, are reviewed highlighting their respective advantages and drawbacks. Lastly, GC × GC techniques are also reported for accurate identification and quantification of terpenes in complex cannabis matrices.
Assuntos
Canabinoides/análise , Cannabis/química , Terpenos/análise , Canabinoides/química , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Extratos Vegetais/química , Terpenos/químicaRESUMO
Monoclonal antibody (mAb) cooperativity is a phenomenon triggered when mAbs couples promote increased bactericidal killing compared to individual partners. Cooperativity has been deeply investigated among mAbs elicited by factor H-binding protein (fHbp), a Neisseria meningitidis surface-exposed lipoprotein and one of the key antigens included in both serogroup B meningococcus vaccine Bexsero and Trumenba. Here we report the structural and functional characterization of two cooperative mAbs pairs isolated from Bexsero vaccines. The 3D electron microscopy structures of the human mAb-fHbp-mAb cooperative complexes indicate that the angle formed between the antigen binding fragments (fAbs) assume regular angle and that fHbp is able to bind simultaneously and stably the cooperative mAbs pairs and human factor H (fH) in vitro. These findings shed light on molecular basis of the antibody-based mechanism of protection driven by simultaneous recognition of the different epitopes of the fHbp and underline that cooperativity is crucial in vaccine efficacy.
Assuntos
Anticorpos Monoclonais/química , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Anticorpos Monoclonais/imunologia , Atividade Bactericida do Sangue , Fator H do Complemento/metabolismo , Mapeamento de Epitopos , Humanos , Vacinas Meningocócicas/imunologia , Microscopia Eletrônica de Transmissão , Ressonância de Plasmônio de SuperfícieRESUMO
Group B Streptococcus (GBS) colonizes the human lower intestinal and genital tracts and constitutes a major threat to neonates from pregnant carrier mothers and to adults with underlying morbidity. The pathogen expresses cell-surface virulence factors that enable cell adhesion and penetration and that counteract innate and adaptive immune responses. Among these, the complement interfering protein (CIP) was recently described for its capacity to interact with the human C4b ligand and to interfere with the classical- and lectin-complement pathways. In the present study, we provide evidence that CIP can also interact with C3, C3b, and C3d. Immunoassay-based competition experiments showed that binding of CIP to C3d interferes with the interaction between C3d and the complement receptor 2/cluster of differentiation 21 (CR2/CD21) receptor on B cells. By B-cell intracellular signaling assays, CIP was confirmed to down-regulate CR2/CD21-dependent B-cell activation. The CIP domain involved in C3d binding was mapped via hydrogen deuterium exchange-mass spectrometry. The data obtained reveal a new role for this GBS polypeptide at the interface between the innate and adaptive immune responses, adding a new member to the growing list of virulence factors secreted by gram-positive pathogens that incorporate multiple immunomodulatory functions.-Giussani, S., Pietrocola, G., Donnarumma, D., Norais, N., Speziale, P., Fabbrini, M., Margarit, I. The Streptococcus agalactiae complement interfering protein combines multiple complement-inhibitory mechanisms by interacting with both C4 and C3 ligands.
Assuntos
Proteínas de Bactérias/metabolismo , Complemento C3d/antagonistas & inibidores , Complemento C4/antagonistas & inibidores , Streptococcus agalactiae/patogenicidade , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proteínas de Bactérias/farmacologia , Sítios de Ligação , Sinalização do Cálcio , Linhagem Celular Tumoral , Complemento C3b/antagonistas & inibidores , Complemento C3b/metabolismo , Complemento C3d/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Ativação Linfocitária/efeitos dos fármacos , Espectrometria de Massas , Ligação Proteica , Mapeamento de Interação de Proteínas , Receptores de Complemento 3d/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Streptococcus agalactiae/imunologia , Streptococcus agalactiae/metabolismo , Ressonância de Plasmônio de Superfície , Virulência , Fatores de Virulência/farmacologiaRESUMO
Hydrogen-deuterium exchange (HDx) associated with mass spectrometry (MS) is emerging as a powerful tool to provide conformational information about membrane proteins. Unfortunately, as for X-ray diffraction and NMR, HDx performed on reconstituted in vitro systems might not always reflect the in vivo environment. Outer-membrane vesicles naturally released by Escherichia coli were used to carry out analysis of native OmpF through HDx-MS. A new protocol compatible with HDx analysis that avoids hindrance from the lipid contents was setup. The extent of deuterium incorporation was in good agreement with the X-ray diffraction data of OmpF as the buried ß-barrels incorporated a low amount of deuterium, whereas the internal loop L3 and the external loops incorporated a higher amount of deuterium. Moreover, the kinetics of incorporation clearly highlights that peptides segregate well in two distinct groups based exclusively on a trimeric organization of OmpF in the membrane: peptides presenting fast kinetics of labeling are facing the complex surrounding environment, whereas those presenting slow kinetics are located in the buried core of the trimer. The data show that HDx-MS applied to a complex biological system is able to reveal solvent accessibility and spatial arrangement of an integral outer-membrane protein complex.
Assuntos
Proteínas de Bactérias/química , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Porinas/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Cinética , Conformação ProteicaRESUMO
Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and organ transplant rejection. The HCMV gH/gL/UL128/UL130/UL131A complex (Pentamer) is the main target of humoral responses and thus a key vaccine candidate. We report two structures of Pentamer bound to human neutralizing antibodies, 8I21 and 9I6, at 3.0 and 5.9 Å resolution, respectively. The HCMV gH/gL architecture is similar to that of Epstein-Barr virus (EBV) except for amino-terminal extensions on both subunits. The extension of gL forms a subdomain composed of a three-helix bundle and a ß hairpin that acts as a docking site for UL128/UL130/UL131A. Structural analysis reveals that Pentamer is a flexible molecule, and suggests sites for engineering stabilizing mutations. We also identify immunogenic surfaces important for cellular interactions by epitope mapping and functional assays. These results can guide the development of effective vaccines and immunotherapeutics against HCMV.
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We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Mapeamento de Epitopos/métodos , Biblioteca de Peptídeos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Reações Cruzadas , Sequenciamento de Nucleotídeos em Larga Escala , Espectrometria de Massas/métodos , Camundongos , Neisseria meningitidis Sorogrupo B/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Aderência Bacteriana/efeitos dos fármacos , Células Epiteliais/microbiologia , Proteínas de Fímbrias/imunologia , Fímbrias Bacterianas/imunologia , Streptococcus pneumoniae/imunologia , Linhagem Celular , Mapeamento de Epitopos , Humanos , Fatores de Virulência/imunologiaRESUMO
Vaccines are the most effective way to fight infectious diseases saving countless lives since their introduction. Their evolution during the last century made use of the best technologies available to continuously increase their efficacy and safety. Mass spectrometry (MS) and proteomics are already playing a central role in the identification and characterization of novel antigens. Over the last years, we have been witnessing the emergence of structural proteomics in vaccinology, as a major tool for vaccine candidate discovery, antigen design and life cycle management of existing products. In this review, we describe the MS techniques associated to structural proteomics and we illustrate the contribution of structural proteomics to vaccinology discussing potential applications.
Assuntos
Proteômica/métodos , Vacinas/química , Animais , Antígenos/química , Antígenos/imunologia , Antígenos/isolamento & purificação , Medição da Troca de Deutério , Mapeamento de Epitopos , Humanos , Espectrometria de Massas , Conformação Proteica , Vacinas/imunologia , Vacinas/isolamento & purificaçãoRESUMO
Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV.
Assuntos
Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Citomegalovirus/imunologia , Epitopos de Linfócito B/imunologia , Proteínas Virais de Fusão/imunologia , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Linhagem Celular , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Humanos , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em Tandem , Transfecção , Internalização do VírusRESUMO
Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Neisseria meningitidis/metabolismo , Ubiquinona/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antimicina A/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Clonagem Molecular , Dissulfetos/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Peróxido de Hidrogênio/farmacologia , Espectrometria de Massas/métodos , Vacinas Meningocócicas/metabolismo , Metacrilatos/farmacologia , Dados de Sequência Molecular , Mutação , Neisseria meningitidis/genética , Neisseria meningitidis/crescimento & desenvolvimento , Oxidantes/farmacologia , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Ligação Proteica , Multimerização Proteica , Tiazóis/farmacologiaRESUMO
Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.