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1.
Microb Cell Fact ; 18(1): 182, 2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31655587

RESUMO

BACKGROUND: Oxidative stress caused by inevitable hostile conditions during fermentative process was the most serious threat to the survival of the well-known industrial microorganism Corynebacterium glutamicum. To survive, C. glutamicum developed several antioxidant defenses including millimolar concentrations of mycothiol (MSH) and protective enzymes. Glutathione (GSH) S-transferases (GSTs) with essentially defensive role in oxidative stress have been well defined in numerous microorganisms, while their physiological and biochemical functions remained elusive in C. glutamicum thus far. RESULTS: In the present study, we described protein NCgl1216 belonging to a novel MSH S-transferase Xi class (MstX), considered as the equivalent of GST Xi class (GSTX). MstX had a characteristic conserved catalytic motif (Cys-Pro-Trp-Ala, C-P-W-A). MstX was active as thiol transferase, dehydroascorbate reductase, mycothiolyl-hydroquinone reductase and MSH peroxidase, while it showed null activity toward canonical GSTs substrate as 1-chloro-2,4-dinitrobenzene (CDNB) and GST Omega's specific substance glutathionyl-acetophenones, indicating MstX had some biochemical characteristics related with mycoredoxin (Mrx). Site-directed mutagenesis showed that, among the two cysteine residues of the molecule, only the residue at position 67 was required for the activity. Moreover, the residues adjacent to the active Cys67 were also important for activity. These results indicated that the thiol transferase of MstX operated through a monothiol mechanism. In addition, we found MstX played important role in various stress resistance. The lack of C. glutamicum mstX gene resulted in significant growth inhibition and increased sensitivity under adverse stress condition. The mstX expression was induced by stress. CONCLUSION: Corynebacterium glutamicum MstX might be critically involved in response to oxidative conditions, thereby giving new insight in how C. glutamicum survived oxidative stressful conditions.


Assuntos
Proteínas de Bactérias/química , Corynebacterium glutamicum/metabolismo , Cisteína/metabolismo , Glutationa Transferase/química , Glicopeptídeos/metabolismo , Inositol/metabolismo , Oxirredução , Estresse Oxidativo
2.
Int J Mol Sci ; 20(19)2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31581426

RESUMO

The advent of multidrug resistance among pathogenic bacteria has attracted great attention worldwide. As a response to this growing challenge, diverse studies have focused on the development of novel anti-infective therapies, including antimicrobial peptides (AMPs). The biological properties of this class of antimicrobials have been thoroughly investigated, and membranolytic activities are the most reported mechanisms by which AMPs kill bacteria. Nevertheless, an increasing number of works have pointed to a different direction, in which AMPs are seen to be capable of displaying non-lytic modes of action by internalizing bacterial cells. In this context, this review focused on the description of the in vitro and in vivo antibacterial and antibiofilm activities of non-lytic AMPs, including indolicidin, buforin II PR-39, bactenecins, apidaecin, and drosocin, also shedding light on how AMPs interact with and further translocate through bacterial membranes to act on intracellular targets, including DNA, RNA, cell wall and protein synthesis.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/metabolismo , Glicopeptídeos/metabolismo , Insetos , Biossíntese de Proteínas
3.
Int J Med Microbiol ; 309(6): 151332, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31350128

RESUMO

Peptidoglycan (PG) is a bacteria specific cell surface layer that ensures the bacterial shape and integrity. The two actinomycetes Amycolatopsis balhimycina and Microbispora sp. PTA-5024 are producers of PG targeting antibiotics. To prevent the binding of their secreted product to their own PG, they developed specific self-resistance mechanisms. Modifications of PG, which are applied by both strains, are the introduction of amide-residues at the PG precursors and the alternative crosslinks within the nascent PG. The PG modifications found in Microbispora sp. PTA-5024 seemed to be an intrinsic characteristic of the genus Microbispora, rather than a specific mechanism of NAI-107 resistance. In contrast, the modifications in A. balhimycina represent an alternative way to avoid suicide specific for glycopeptide producers. The different PG modifications reflect the fact that antibiotic producing organisms contain not only one but multiple mechanisms to ensure protection against biologically active molecules produced by themselves.


Assuntos
Actinobacteria/fisiologia , Antibacterianos/metabolismo , Farmacorresistência Bacteriana , Peptidoglicano/biossíntese , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glicopeptídeos/metabolismo , Peptidoglicano/química , Polimerização
4.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 6): 405-411, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204686

RESUMO

Neutral endopeptidase (neprilysin; NEP) is a proteinase that cleaves a wide variety of peptides and has been implicated in Alzheimer's disease, cardiovascular conditions, arthritis and other inflammatory diseases. The structure of the soluble extracellular domain (residues 55-750) of rabbit neprilysin was solved both in its native form at 2.1 Šresolution, and bound to the inhibitors phosphoramidon and thiorphan at 2.8 and 3.0 Šresolution, respectively. Consistent with the extracellular domain of human neprilysin, the structure reveals a large central cavity which contains the active site and the location for inhibitor binding.


Assuntos
Glicopeptídeos/metabolismo , Modelos Moleculares , Neprilisina/química , Neprilisina/metabolismo , Inibidores de Proteases/metabolismo , Tiorfano/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Glicopeptídeos/química , Inibidores de Proteases/química , Conformação Proteica , Coelhos , Especificidade por Substrato , Tiorfano/química
5.
Biochim Biophys Acta Biomembr ; 1861(8): 1510-1521, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226245

RESUMO

Hassallidins are cyclic glycolipopeptides produced by cyanobacteria and other prokaryotes. The hassallidin structure consists of a peptide ring of eight amino acids where a fatty acid chain, additional amino acids, and sugar moieties are attached. Hassallidins show antifungal activity against several opportunistic human pathogenic fungi, but does not harbor antibacterial effects. However, they have not been studied on mammalian cells, and the mechanism of action is unknown. We purified hassallidin D from cultured cyanobacterium Anabaena sp. UHCC 0258 and characterized its effect on mammalian and fungal cells. Ultrastructural analysis showed that hassallidin D disrupts cell membranes, causing a lytic/necrotic cell death with rapid presence of disintegrated outer membrane, accompanied by internalization of small molecules such as propidium iodide into the cells. Furthermore, artificial liposomal membrane assay showed that hassallidin D selectively targets sterol-containing membranes. Finally, in silico membrane modeling allowed us to study the interaction between hassallidin D and membranes in detail, and confirm the role of cholesterol for hassallidin-insertion into the membrane. This study demonstrates the mechanism of action of the natural compound hassallidin, and gives further insight into how bioactive lipopeptide metabolites selectively target eukaryotic cell membranes.


Assuntos
Antifúngicos/metabolismo , Antineoplásicos/metabolismo , Glicolipídeos/metabolismo , Glicopeptídeos/metabolismo , Lipopeptídeos/metabolismo , Lipídeos de Membrana/metabolismo , Esteróis/metabolismo , Anabaena/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Glicopeptídeos/isolamento & purificação , Glicopeptídeos/farmacologia , Humanos , Lipopeptídeos/isolamento & purificação , Lipopeptídeos/farmacologia , Membranas Mitocondriais/efeitos dos fármacos
6.
Tuberculosis (Edinb) ; 116: 44-55, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31153518

RESUMO

Low molecular weight (LMW) thiols are molecules with a functional sulfhydryl group that enable them to detoxify reactive oxygen species, reactive nitrogen species and other free radicals. Their roles range from their ability to modulate the immune system to their ability to prevent damage of biological molecules such as DNA and proteins by protecting against oxidative, nitrosative and acidic stress. LMW thiols are synthesized and found in both eukaryotes and prokaryotes. Due to their beneficial role to both eukaryotes and prokaryotes, their specific functions need to be elucidated, most especially in pathogenic prokaryotes such as Mycobacterium tuberculosis (M.tb), in order to provide a rationale for targeting their biosynthesis for drug development. Ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC) are LMW thiols that have been shown to interplay to protect M.tb against cellular stress. Though ERG, MSH and GGC seem to have overlapping functions, studies are gradually revealing their unique physiological roles. Understanding their unique physiological role during the course of tuberculosis (TB) infection, would pave the way for the development of drugs that target their biosynthetic pathway. This review identifies the knowledge gap in the unique physiological roles of LMW thiols and proposes their mechanistic roles based on previous studies. In addition, it gives an update on identified inhibitors of their biosynthetic enzymes.


Assuntos
Mycobacterium tuberculosis/metabolismo , Compostos de Sulfidrila/metabolismo , Tuberculose/microbiologia , Animais , Antituberculosos/uso terapêutico , Cisteína/metabolismo , Dipeptídeos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Enzimas/metabolismo , Ergotioneína/metabolismo , Glicopeptídeos/metabolismo , Humanos , Inositol/metabolismo , Terapia de Alvo Molecular , Peso Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Tuberculose/tratamento farmacológico
7.
Carbohydr Res ; 480: 54-60, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31176190

RESUMO

Often glycosidase assays are based on small-molecule compounds where a glycan of interest is linked to a chromophore allowing for easy detection of cleavage of the glycoside bond. However, such compounds only resemble part of the more complex substrate molecule for enzymes acting on glycoconjugates of glycopeptides or glycoproteins. Nonetheless, the advantage is obvious as enzyme activity is readily recorded and kinetic parameters easily obtained. This is not often the case with glycopeptides or glycoproteins as these may reveal increased complexity in terms of heterogeneity in protein-glycan stoichiometry and restricted enzyme accessibility. However, a kinetic analysis of glycan release from glycopeptides could provide information complementary to that of small-molecule substrates, especially if providing kinetic parameters that are immediately comparable. We have characterized the steady state kinetics of wild type and mutant variants of Bifidobacterium longum endo-α-N-acetylgalactosaminidase, by recording the enzymatic release of Galß(1-3)GalNAc from bovine glycomacropeptide pre-treated with sialidase to remove sialic acid units. Differences between previously reported kinetic constants obtained with synthetic substrates and those obtained in the present work demonstrate an influence of the peptide moiety on the kinetic properties of endo-α-N-acetylgalactosaminidase. The devised assay and data handling method determines the accessible substrate concentration as well as the steady state kinetic parameters, KM and kcat, for glycoconjugates of glycopeptides described by the same units as obtained from using small-molecule substrates and thus allows for a direct comparison.


Assuntos
Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Biocatálise , Polissacarídeos/química , alfa-N-Acetilgalactosaminidase/metabolismo , Bifidobacterium longum/enzimologia , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Cinética , Especificidade por Substrato
8.
BMC Med ; 17(1): 85, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31035998

RESUMO

INTRODUCTION: Copeptin is the stable surrogate marker of vasopressin (VP), which is released in response to elevated plasma osmolality or low blood pressure. Elevated plasma copeptin levels are associated with higher risk of insulin resistance-related disorders, such as type 2 diabetes (T2DM), metabolic syndrome (MS), and cardiovascular disease, and experimental reduction of circulating VP levels is shown to significantly decrease hepatic fat content in obese rats, independently from body adiposity. However, the association between copeptin and non-alcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) in humans has not been explored yet. The aim of this study was to explore the relationship between plasma copeptin and the presence/severity of NAFLD/NASH. METHODS: For this study, we recruited 60 obese patients candidate to bariatric surgery for clinical purposes in which intraoperative liver biopsies were performed for diagnosing NAFLD/NASH. Circulating copeptin levels were also assessed in 60 age- and sex-comparable non-obese individuals without NAFLD at liver ultrasonography. Plasma copeptin was measured by sandwich immunoluminometric assay (Thermo Fisher Scientific). RESULTS: Obese patients with biopsy-proven NAFLD (53%) had significantly higher copeptin levels than both obese individuals without NAFLD and non-obese subjects (ob/NAFLD+ 9.5 ± 4.9; ob/NAFLD- 6.4 ± 2.6; and non-ob/NAFLD- 7.4 ± 5.1 pmol/L; p = 0.004 and p = 0.01 respectively). Plasma copeptin concentration positively correlated with hepatic macro- and micro-vesicular steatosis (r = 0.36, p = 0.026; r = 0.31, p = 0.05), lobular inflammation (r = 0.37, p = 0.024) and significantly increased throughout degrees of NASH severity, as expressed as absence, borderline, and overt NASH at the liver biopsy (r = 0.35, p = 0.01). Greater circulating copeptin predicted the presence of NASH with OR = 1.73 (95% CI = 1.02-2.93) after multivariate adjustment for age, sex, renal function and presence of T2DM and MS components. CONCLUSIONS: Increased plasma copeptin is independently associated with the presence and severity of NAFLD and NASH, pointing to a novel mechanism behind human fatty liver disease potentially modifiable by pharmacological treatment and lifestyle intervention.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Glicopeptídeos/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade/complicações , Adulto , Animais , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia
9.
Mol Cell Proteomics ; 18(7): 1396-1409, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31040225

RESUMO

Most proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in coordinated, but poorly understood ways - guided partly by the kinetic properties and substrate specificities of their catalytic domains, as well as by modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of nonredundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cells with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, whereas distinct O-glycosite subsets are covered by nonredundant GalNAc-T isoform-specific functions. We demonstrate that the GalNAc-T7 and T10 isoforms function in follow-up of high-density O-glycosylated regions, and that GalNAc-T11 has highly restricted functions and essentially only serves the low-density lipoprotein-related receptors in linker regions (C6XXXTC1) between the ligand-binding repeats.


Assuntos
Glicômica , Proteômica , Glicopeptídeos/metabolismo , Glicosilação , Células HEK293 , Células Hep G2 , Humanos , Proteoma/metabolismo
10.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022913

RESUMO

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the enzyme iduronate 2-sulfatase (IDS), which leads to the accumulation of glycosaminoglycans in most organ-systems, including the brain, and resulting in neurological involvement in about two-thirds of the patients. The main treatment is represented by a weekly infusion of the functional enzyme, which cannot cross the blood-brain barrier and reach the central nervous system. In this study, a tailored nanomedicine approach based on brain-targeted polymeric nanoparticles (g7-NPs), loaded with the therapeutic enzyme, was exploited. Fibroblasts from MPSII patients were treated for 7 days with NPs loaded with the IDS enzyme; an induced IDS activity like the one detected in healthy cells was measured, together with a reduction of GAG content to non-pathological levels. An in vivo short-term study in MPSII mice was performed by weekly administration of g7-NPs-IDS. Biochemical, histological, and immunohistochemical evaluations of liver and brain were performed. The 6-weeks treatment produced a significant reduction of GAG deposits in liver and brain tissues, as well as a reduction of some neurological and inflammatory markers (i.e., LAMP2, CD68, GFAP), highlighting a general improvement of the brain pathology. The g7-NPs-IDS approach allowed a brain-targeted enzyme replacement therapy. Based on these positive results, the future aim will be to optimize NP formulation further to gain a higher efficacy of the proposed approach.


Assuntos
Encéfalo/efeitos dos fármacos , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Iduronato Sulfatase/administração & dosagem , Mucopolissacaridose II/tratamento farmacológico , Nanopartículas/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Encéfalo/patologia , Portadores de Fármacos/química , Terapia de Reposição de Enzimas , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Humanos , Iduronato Sulfatase/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucopolissacaridose II/enzimologia , Mucopolissacaridose II/metabolismo , Mucopolissacaridose II/patologia , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química
11.
Anal Bioanal Chem ; 411(18): 4175-4183, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31020367

RESUMO

In this work, the chemiluminescence (CL) property of 5,10,15,20-tetrakis(4-carboxyphenyl)-porphyrin- and N-(4-aminobutyl)-N-ethylisoluminol-functionalized TiO2 nanoparticles (TiO2-TCPP-ABEI nanoluminophores) was studied for the first time. It was found that TiO2-TCPP-ABEI nanoluminophores exhibited excellent CL activity in the presence of H2O2. The CL mechanism has been proposed due to the reaction of ABEI with H2O2 and catalytic effect of TiO2 and TCPP. Furthermore, trisodium citrate-stabilized gold nanoparticles were observed to effectively quench the CL of TiO2-TCPP-ABEI due to CL resonance energy transfer (CRET). On this basis, a sensitive and selective CRET-based immunoassay was developed for the determination of copeptin by using TiO2-TCPP-ABEI nanoluminophores as both CL nanointerface and energy donor, and using cit-AuNPs as an effective energy receptor. The immunoassay exhibited a wide dynamic range from 5 × 10-12 to 1 × 10-9 g mL-1 with a low detection limit of 1.54 × 10-12 g mL-1, which was superior to previously reported CL-based immunoassays. It was successfully applied for the determination of copeptin in serum samples, which would provide a good practical perspective on the clinical diagnosis. This strategy may also be used for the detection of other antigens if corresponding antibodies are available. Graphical abstract.


Assuntos
Transferência de Energia , Glicopeptídeos/metabolismo , Cardiopatias/metabolismo , Luminescência , Luminol/análogos & derivados , Nanopartículas Metálicas/química , Titânio/química , Biomarcadores/análise , Humanos , Luminol/química
12.
Talanta ; 199: 254-261, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952254

RESUMO

N-glycosylation is deeply involved in many biological processes, and approximately 50% of mammalian proteins are predicted to be glycosylated. Many large-scale studies have been carried out to reveal the glycosylation status involved in different physiological pathologies across species. However, the lack of a highly specific and high-throughput N-glycosylated enrichment method not only results in extended time requirements but also limits the depth of mapping when handling a large number of samples. In this study, we firstly optimized traditional zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) enrichment and found that using of 70% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) as the enrichment buffer, 2800 g as the washing speed and 600 µL as the washing volume achieved the best specificity, which is higher than 75%. On this basis, we developed a multi-parallel enrichment strategy assisted by a filter-coated 96-well plate, which achieved high specificity and high throughput simultaneously. This strategy allowed us to enrich large numbers of fractionated samples from hepatocellular carcinoma (HCC) cell lines in less than 2 h. Its good specificity helped us achieve in-depth mapping of the N-glycoproteome in metastatic HCC cell lines. A total of 5466 N-glycosites from 2383 glycoproteins were identified, among which 1900 N-glycosites were unannotated in UniProt. The in-depth glycoproteome mapping provides insight into the N-glycosylation status in HCC cell lines with differences in metastatic potential and contributes to biomarker discovery.


Assuntos
Carcinoma Hepatocelular/química , Glicopeptídeos/química , Ensaios de Triagem em Larga Escala , Neoplasias Hepáticas/química , Proteoma/análise , Carcinoma Hepatocelular/metabolismo , Cromatografia Líquida , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Neoplasias Hepáticas/metabolismo , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Células Tumorais Cultivadas
13.
Arch Microbiol ; 201(6): 713-722, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30868174

RESUMO

The genus Pantoea contains a broad range of plant-associated bacteria, including some economically important plant pathogens as well as some beneficial members effective as biological control agents of plant pathogens. The most well-characterized representatives of biological control agents from this genus generally produce one or more antimicrobial compounds adding to biocontrol efficacy. Some Pantoea species evaluated as biocontrol agents for fire blight disease of apple and pear produce a histidine-reversible antibiotic. Three commonly studied histidine-reversible antibiotics produced by Pantoea spp. are herbicolin O, MccEh252, and pantocin A. Pantocin A is a novel ribosomally encoded and post-translationally modified peptide natural product. Here, we review the current knowledge on the chemistry, genetics, biosynthesis, and incidence and environmental relevance of pantocin A and related histidine-reversible antibiotics produced by Pantoea.


Assuntos
Agentes de Controle Biológico/metabolismo , Glicopeptídeos/metabolismo , Pantoea/metabolismo , Peptídeos/metabolismo , Doenças das Plantas/microbiologia , Agentes de Controle Biológico/química , Agentes de Controle Biológico/farmacologia , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Pantoea/química , Pantoea/genética , Peptídeos/química , Peptídeos/farmacologia
14.
Molecules ; 24(6)2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30871155

RESUMO

The initial contact of pathogens with host cells is usually mediated by their adhesion to glycan structures present on the cell surface in order to enable infection. Furthermore, glycans play important roles in the modulation of the host immune responses to infection. Understanding the carbohydrate-pathogen interactions are of importance for the development of novel and efficient strategies to either prevent, or interfere with pathogenic infection. Synthetic glycopeptides and mimetics thereof are capable of imitating the multivalent display of carbohydrates at the cell surface, which have become an important objective of research over the last decade. Glycopeptide based constructs may function as vaccines or anti-adhesive agents that interfere with the ability of pathogens to adhere to the host cell glycans and thus possess the potential to improve or replace treatments that suffer from resistance. Additionally, synthetic glycopeptides are used as tools for epitope mapping of antibodies directed against structures present on various pathogens and have become important to improve serodiagnostic methods and to develop novel epitope-based vaccines. This review will provide an overview of the most recent advances in the synthesis and application of glycopeptides and glycopeptide mimetics exhibiting a peptide-like backbone in glycobiology.


Assuntos
Infecções Bacterianas/imunologia , Glicopeptídeos/metabolismo , Viroses/imunologia , Animais , Anticorpos/química , Anticorpos/farmacologia , Materiais Biomiméticos/farmacologia , Epitopos/química , Glicosilação , Humanos , Vacinas/farmacologia
15.
Anal Chim Acta ; 1053: 43-53, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30712568

RESUMO

Hydrophilic interaction chromatography (HILIC) utilizing zwitterion-modified material as solid phase has attracted extensive attention for selective enrichment of glycopeptides. However, a tedious synthesis and low specificity for glycopeptides have restricted its application. Herein, a facile and effective approach was developed to synthesize a zwitterionic (ZIC) polymer-coated magnetic composites (denoted as Fe3O4@PMSA) with a zwitterion ((2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide, MSA) via one-step distillation-precipitation polymerization (DPP). The well-designed composites presented clearly ZIC-polymer shell and superior hydrophilicity (water contact angle 30.2), and the performance for selective enrichment of glycopeptides were investigated with standard and real samples, respectively. Owning to the abundant of ZIC molecules with multi-charge and polar groups on the surface of resulting polymer coating, the Fe3O4@PMSA demonstrated high selectivity for glycopeptides enrichment with IgG digest (twenty glycopeptides identified, S/N ≥ 20) and a mixture of IgG and BSA at the mass ratio of 1:230 (sixteen glycopeptides identified, S/N ≥ 20). Besides, the detection limit as low as 0.67 fmol for IgG (S/N ≥ 10) and satisfied recovery yield more than 74% were achieved by the proposed sorbents. Finally, the Fe3O4@PMSA was applied for enriching N-linked glycopeptides from human serum, and 348 unique N-glycosylation sites and 419 glycopeptides from 158 glycoproteins were strictly identified from 1 µL human serum. The results demonstrated that the proposed Fe3O4@PMSA showed a promising potential in glycoproteomics analysis of real biological samples.


Assuntos
Glicopeptídeos/química , Glicopeptídeos/isolamento & purificação , Polimerização , Precipitação Química , Óxido Ferroso-Férrico/química , Glicopeptídeos/metabolismo , Proteólise , Tripsina/metabolismo
16.
Biochemistry ; 58(8): 1131-1140, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30694662

RESUMO

Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)-carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-ß-d- N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new "glycoamino acid" metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-ß-d- N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.


Assuntos
Aminoácidos/química , Vias Biossintéticas , Carboidratos/química , Glicopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeo Sintases/metabolismo , Photorhabdus/metabolismo , Glicopeptídeos/química , Família Multigênica , Fragmentos de Peptídeos/química , Peptídeo Sintases/genética , Photorhabdus/genética , Photorhabdus/crescimento & desenvolvimento
17.
Artigo em Inglês | MEDLINE | ID: mdl-30323040

RESUMO

Occidiofungin is produced by the soil bacterium Burkolderia contaminans MS14 and is structurally similar or identical to the burkholdines, xylocandins, and cepacidines. This study identified the primary cellular target of occidiofungin, which was determined to be actin. The modification of occidiofungin with a functional alkyne group enabled affinity purification assays and localization studies in yeast. Occidiofungin has a subtle effect on actin dynamics that triggers apoptotic cell death. We demonstrate the highly specific localization of occidiofungin to cellular regions rich in actin in yeast and the binding of occidiofungin to purified actin in vitro Furthermore, a disruption of actin-mediated cellular processes, such as endocytosis, nuclear segregation, and hyphal formation, was observed. All of these processes require the formation of stable actin cables, which are disrupted following the addition of a subinhibitory concentration of occidiofungin. We were also able to demonstrate the effectiveness of occidiofungin in treating a vulvovaginal yeast infection in a murine model. The results of this study are important for the development of an efficacious novel class of actin binding drugs that may fill the existing gap in treatment options for fungal infections or different types of cancer.


Assuntos
Actinas/metabolismo , Antifúngicos/uso terapêutico , Burkholderia/metabolismo , Candidíase Vulvovaginal/tratamento farmacológico , Glicopeptídeos/metabolismo , Glicopeptídeos/uso terapêutico , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/uso terapêutico , Animais , Candida/efeitos dos fármacos , Feminino , Glicopeptídeos/química , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos Cíclicos/química
18.
Talanta ; 191: 509-518, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30262092

RESUMO

It is challenging to capture N-glycopeptides with high recovery and high specificity from complicated biosystems. Herein, we present a facile and economical procedure to generate a novel self-assembling 4-Mercaptobenzene boronic acid functionalized and Au-doped Straticulate C3N4 (MASC), with enhanced affinity capability towards glycopeptides. The materials possess low pH value adaptation, high hydrophilicity and stability, good repeatability and recyclability, and provided high selectivity (1:100), low limit of detection (0.33 fmol/µL), high enrichment efficiency (~ 80%) and high recovery rate (~ 90%) towards glycopeptides. The materials can capture glycopeptides unbiasedly, as demonstrated by the identification of 37 glycopeptides from IgG and 21 glycopeptides from horseradish peroxidase (HRP). The performance of MASC on human urine and serum glycoproteome analysis was also tested. An average of 1465 glycopeptides from 839 glycoproteins and 1553 glycopeptides from 884 glycoproteins were identified from female and male urine samples in a single mass spectrometry analysis. O-glycopeptides from human urine were also significantly enriched. Additionally, 463 glycopeptides assigned to 209 glycoproteins were identified from 5 µL of human serum. All of these results indicate that MASC presents a good performance and applicability in the field of glycoproteomic research.


Assuntos
Ácidos Borônicos/química , Glicopeptídeos/química , Interações Hidrofóbicas e Hidrofílicas , Nitrilos/química , Feminino , Glicopeptídeos/metabolismo , Glicopeptídeos/urina , Humanos , Masculino , Modelos Moleculares , Conformação Molecular , Caracteres Sexuais , Tripsina/metabolismo
19.
Low Urin Tract Symptoms ; 11(1): 43-47, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29057582

RESUMO

OBJECTIVE: The aim of the present study was to investigate the use of random copeptin concentrations as possible biomarkers for the differential diagnosis of nocturnal polyuria (NP). METHODS: In all, 111 patients with and without nocturia were enrolled in the study. Patients with a neurogenic bladder and/or those who had undergone bladder or urethral surgery were excluded from the study. All patients completed a 72-hour frequency-volume chart and a renal function profile. A random blood sample was obtained during the day for measurement of plasma copeptin concentrations, osmolality, and serum sodium and creatinine concentrations. The effect of the use of different definitions for NP was evaluated. RESULTS: The median age of the study participants was 61 years, and 48% were female. Copeptin was significantly correlated with urinary and plasma osmolality, as well as free water clearance (r=0.43, 0.56 and -0.38 respectively; P < .001 for all). Study participants were divided into 3 groups: controls (n = 51), those with NP (n = 41), and those with global polyuria (n = 19). Copeptin concentrations were significantly lower in subjects with global polyuria than in those with NP and the control group (2.96 vs 3.97 and 3.94 pM, respectively; P = .008 and .005). There was no significant difference in random daytime copeptin concentrations between the NP and control groups (P = .972). The results differed when other definitions for NP were used (e.g. NPi33 or NUP10). CONCLUSIONS: We could not confirm our hypothesis that patients with NP have lower copeptin concentrations, although random blood sampling is not ideal. Further research is required to determine the use of copeptin in NP, perhaps in the identification of the desmopressin response.


Assuntos
Glicopeptídeos/metabolismo , Noctúria/diagnóstico , Poliúria/diagnóstico , Medicina de Precisão/métodos , Idoso , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Caracteres Sexuais
20.
Stress ; 22(1): 93-102, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30369292

RESUMO

The central noradrenaline (NA) stress-response network co-mediates hypothalamic-pituitary-adrenal (HPA) axis activation and arginine-vasopressin (AVP) release. Dysregulation of these systems contributes to stress-related diseases such as human obesity, but their interrelation remains unclear. The study was aimed to test for the first time in vivo whether central noradrenergic activity quantitatively indexed by the availability of the presynaptic NA transporter (NAT) is associated with HPA axis responsiveness as measured with the combined dexamethasone suppression/corticotropin releasing hormone stimulation (dex/CRH) test and copeptin as a surrogate marker of the serum AVP tone in highly obese, otherwise, healthy individuals compared to age- and sex-matched non-obese, healthy controls. In order to assess central NAT availability, positron emission tomography (PET) was applied using the NAT-selective radiotracer S,S-[11C]O-methylreboxetine (MRB) and correlated with curve indicators derived from the dex/CRH test (maximum, MAX, and area under the curve, AUC, for cortisol and adrenocorticotropic hormone, ACTH) as well as with copeptin. In non-obese controls, positive correlations were found between the NAT distribution volume ratios (DVR) of the orbitofrontal cortex (OFC) and the amygdala with the HPA response (OFC: ACTHMAX r = 0.87, p = .001; cortisolMAX r = 0.86, p = .002; amygdala: ACTHMAX r = 0.86, p = .002; cortisolMAX r = 0.79, p = .006), while in obesity, the hypothalamic DVR correlated inversely with the HPA axis response (cortisolMAX, r = -0.66, p = .04) and with copeptin (r = -0.71, p = .02). This association of central NAT availability with HPA axis responsiveness and copeptin suggests a mechanistic interaction between noradrenergic transmission with HPA axis activity and the serum AVP system that differs between non-obese individuals with prefrontal-limbic involvement and obesity with a hypothalamic-centered relationship. Whether the latter finding contributes to obesogenic behavior needs to be further explored.


Assuntos
Glicopeptídeos/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Obesidade/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/sangue , Adulto , Arginina Vasopressina/metabolismo , Encéfalo/metabolismo , Hormônio Liberador da Corticotropina , Dexametasona/farmacologia , Feminino , Glicopeptídeos/sangue , Humanos , Hidrocortisona/sangue , Hidrocortisona/metabolismo , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Masculino , Norepinefrina/metabolismo , Obesidade/sangue , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Estresse Psicológico
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