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1.
Chemosphere ; 256: 127099, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32470733

RESUMO

Membrane fouling is an issue of concern due to the hydrophobic properties of polyethersulfone (PES) membrane when applied in water treatment. In this work, a facile hydrothermal method was utilized to synthesize hierarchical flower-like structured molybdenum disulfide nanosheets (HF-MoS2 NSs) that then incorporated into PES membranes as composite membranes. We characterized their permeability, the separation performance, the antifouling performance, and the antibacterial activity systematically. Results showed that composite membranes exhibited a better pure water flux (286 LMH/bar) at the HF-MoS2 NSs content of 0.4 wt%, which was 1.8 times higher than the control membrane. Also, composite PES membranes achieved 98.2% and 96.9% rejection of BSA and HA in comparison with the control PES membrane (87.3%, and 84.5%, respectively). Compare to the control PES membrane, the flux recovery ratio of the composite membrane increased from 69% to 88% for BSA fouling and increased from 84% to 93% for HA fouling. The retention rate for the organic dyes also improved slightly after HF-MoS2 NSs incorporation into the membrane. Additionally, the composite membranes exhibited a relatively high antibacterial activity against E. coli and B. subtilis with antibacterial rates of 67.8% and 82.5%, respectively. In conclusion, HF-MoS2 NSs incorporated composite membranes were shown to have outstanding filtration performance and could be a promising candidate for practical application in water filtration.


Assuntos
Nanoestruturas/química , Polímeros/química , Sulfonas/química , Purificação da Água/métodos , Dissulfetos/química , Escherichia coli , Filtração , Interações Hidrofóbicas e Hidrofílicas , Membranas Artificiais , Molibdênio/química , Permeabilidade , Água/química
2.
Ecotoxicol Environ Saf ; 200: 110772, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32464444

RESUMO

Recently two-dimensional nanomaterials, such as graphene and molybdenum disulfide (MoS2), have received much attention as adsorbent materials for the effective removal of organic contaminants. MoS2 is attracting attention, not only for its chemical-physical properties, but also for its wide availability in nature as a constituent of molybdenite. The aim of this investigation was to assess the effects of different MoS2 concentrations (5 × 10-1, 5 × 10-2 and 5 × 10-3 mg/ml) on the embryonated eggs of Gallus gallus domesticus, according to Beck method. We evaluated the toxic effect of the MoS2 powder purchased at Sigma-Aldrich indicated as "received" and MoS2 powder treated via mechanical milling indicated as "ball mille". Subsequently, the embryos were sacrificed at different times of embryonic development (11th, 15th and 19th day after incubation) in order to evaluate their embryotoxic and teratogenic effects. The alterations of the embryonic development were studied by morphological and immunohistochemical analysis of the tissues. The results obtained have shown the toxicity of both powders of MoS2 with a high percentage of deaths and growth delays. Moreover, the immunohistochemical analysis performed on several tissue sections showed a strong positivity to the anti-metallothionein1 antibody only for the erythrocytes.


Assuntos
Dissulfetos/química , Dissulfetos/toxicidade , Desenvolvimento Embrionário/efeitos dos fármacos , Molibdênio/química , Molibdênio/toxicidade , Nanopartículas/química , Nanopartículas/toxicidade , Animais , Embrião de Galinha , Relação Dose-Resposta a Droga , Grafite/química , Coração/efeitos dos fármacos , Coração/embriologia , Fígado/efeitos dos fármacos , Fígado/embriologia , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/embriologia , Pulmão/patologia , Tamanho da Partícula , Propriedades de Superfície
3.
Chemosphere ; 255: 126995, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32416394

RESUMO

In this paper, we present the preparation of MoS2-modified magnetic biochar (MoS2@MBC) as a novel adsorbent by a simple hydrothermal method. MoS2@MBC contains abundant S-containing functional groups that facilitate efficient Cd(II) removal from aqueous systems. We employed various characterization techniques to explore the morphology, surface area, and chemical composition of MoS2@MBC; these included Brunauer-Emmett-Teller analysis scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction,. The results indicated the successful decoration of the surface of MoS2@MBC with iron and MoS2, and a higher surface area of MoS2@MBC than that of unmodified biochar. Moreover, adsorption properties including thermodynamics and kinetics were investigated along with the effects of pH, humic acid, and ionic strength on the Cd(II) adsorption onto MoS2@MBC. The O-, C-, S-, and Fe-containing functional groups on the surface of MoS2@MBC led to an electrostatic attraction of Cd(II) and strong Cd-S complexation. The Langmuir and pseudo second-order models fitted best for the batch adsorption experiments results. The adsorption capacity of MoS2@MBC (139 mg g-1 on the basis of the Langmuir model) was 7.81 times higher than that of pristine biochar. The adsorption process was found to be pH-dependent. The experimental results indicated that MoS2@MBC is an effective adsorbent for removing Cd(II) from water solutions. Further, the adsorption process involved the complexation of Cd(II) with oxygen-based functional groups, ion exchange, electrostatic attraction, Cd(II)-π interactions, metal-sulfur complexation, and inner-surface complexation. This work provides new insights into the Cd(II) ions removal from water via adsorption. It also demonstrates that MoS2@MBC is an efficient and economic adsorbent to treat Cd(II)-contaminated water.


Assuntos
Adsorção , Cádmio/isolamento & purificação , Carvão Vegetal/química , Dissulfetos/química , Molibdênio/química , Purificação da Água/métodos , Cádmio/química , Cinética , Concentração Osmolar , Poluentes Químicos da Água/química , Poluentes Químicos da Água/isolamento & purificação
4.
Int J Nanomedicine ; 15: 2971-2986, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32431496

RESUMO

Background: Due to their extraordinary physical and chemical properties, MoS2 nanosheets (MSNs) are becoming more widely used in nanomedicine. However, their influence on immune systems remains unclear. Materials and Methods: Two few-layered MSNs at sizes of 100-250 nm (S-MSNs) and 400-500 nm (L-MSNs) were used in this study. Bone marrow-derived dendritic cells (DCs) were exposed to both MSNs at different doses (0, 8, 16, 32, 64, 128 µg/mL) for 48 h and subjected to analyses of surface marker expression, cytokine secretion, lymphoid homing and in vivo T cell priming. Results: Different-sized MSNs of all doses did not affect the viability of DCs. The expression of CD40, CD80, CD86 and CCR7 was significantly higher on both S-MSN- and L-MSN-treated DCs at a dose of 128 µg/mL. As the dose of MSN increased, the secretion of IL-12p70 remained unchanged, the secretion of IL-1ß decreased, and the production of TNF-α increased. A significant increase in IL-6 was observed in the 128 µg/mL L-MSN-treated DCs. In particular, MSN treatment dramatically improved the ex vivo movement and in vivo homing ability of both the local resident and blood circulating DCs. Furthermore, the cytoskeleton rearrangement regulated by ROS elevation was responsible for the enhanced homing ability of the MSNs. More robust CD4+ and CD8+ T cell proliferation and activation (characterized by high expression of CD107a, CD69 and ICOS) was observed in mice vaccinated with MSN-treated DCs. Importantly, exposure to MSNs did not interrupt LPS-induced DC activation, homing and T cell priming. Conclusion: Few-layered MSNs ranging from 100 to 500 nm in size could play an immunostimulatory role in enhancing DC maturation, migration and T cell elicitation, making them a good candidate for vaccine adjuvants. Investigation of this study will not only expand the applications of MSNs and other new transition metal dichalcogenides (TMDCs) but also shed light on the in vivo immune-risk evaluation of MSN-based nanomaterials.


Assuntos
Diferenciação Celular , Movimento Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Dissulfetos/farmacologia , Molibdênio/farmacologia , Nanopartículas/química , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Dendríticas/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/efeitos dos fármacos
5.
Nat Commun ; 11(1): 2264, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385283

RESUMO

ABCG2 is an ABC transporter that extrudes a variety of compounds from cells, and presents an obstacle in treating chemotherapy-resistant cancers. Despite recent structural insights, no anticancer drug bound to ABCG2 has been resolved, and the mechanisms of multidrug transport remain obscure. Such a gap of knowledge limits the development of novel compounds that block or evade this critical molecular pump. Here we present single-particle cryo-EM studies of ABCG2 in the apo state, and bound to the three structurally distinct chemotherapeutics. Without the binding of conformation-selective antibody fragments or inhibitors, the resting ABCG2 adopts a closed conformation. Our cryo-EM, biochemical, and functional analyses reveal the binding mode of three chemotherapeutic compounds, demonstrate how these molecules open the closed conformation of the transporter, and establish that imatinib is particularly effective in stabilizing the inward facing conformation of ABCG2. Together these studies reveal the previously unrecognized conformational cycle of ABCG2.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Antineoplásicos/química , Transporte Biológico , Dissulfetos/metabolismo , Células HEK293 , Humanos , Mesilato de Imatinib/metabolismo , Ligantes , Mitoxantrona/química , Mitoxantrona/metabolismo , Modelos Biológicos , Estrutura Secundária de Proteína
6.
PLoS Biol ; 18(4): e3000656, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271748

RESUMO

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here, we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide cross-linking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 ß1-strand, while also recapitulating earlier findings from nuclear magnetic resonance, modeling and crystallography of homologous receptors. A cross-linking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor targeting small molecules and biologics with novel pharmacology.


Assuntos
Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Animais , Sítios de Ligação , Western Blotting , Quimiocina CXCL12/genética , Cisteína/química , Cisteína/genética , Dissulfetos/química , Citometria de Fluxo , Células HEK293 , Humanos , Insetos/citologia , Modelos Moleculares , Mutação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores CXCR4/genética , beta-Arrestinas/metabolismo
7.
Nature ; 580(7802): 216-219, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269349

RESUMO

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.


Assuntos
Hidrolases/química , Hidrolases/metabolismo , Plásticos/química , Plásticos/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Engenharia de Proteínas , Reciclagem , Actinobacteria/enzimologia , Burkholderiales/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Ensaios Enzimáticos , Estabilidade Enzimática , Fusarium/enzimologia , Modelos Moleculares , Ácidos Ftálicos/metabolismo , Polimerização
8.
Nat Commun ; 11(1): 1725, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265442

RESUMO

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.


Assuntos
Glutarredoxinas/química , Glutationa/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos/genética , Catálise , Domínio Catalítico/genética , Dissulfetos/química , Ativação Enzimática , Ensaios Enzimáticos , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/química , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Conformação Proteica em alfa-Hélice , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Nat Commun ; 11(1): 1814, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286284

RESUMO

X-ray free-electron lasers (XFELs) enable crystallographic structure determination beyond the limitations imposed upon synchrotron measurements by radiation damage. The need for very short XFEL pulses is relieved through gating of Bragg diffraction by loss of crystalline order as damage progresses, but not if ionization events are spatially non-uniform due to underlying elemental distributions, as in biological samples. Indeed, correlated movements of iron and sulfur ions were observed in XFEL-irradiated ferredoxin microcrystals using unusually long pulses of 80 fs. Here, we report a femtosecond time-resolved X-ray pump/X-ray probe experiment on protein nanocrystals. We observe changes in the protein backbone and aromatic residues as well as disulfide bridges. Simulations show that the latter's correlated structural dynamics are much slower than expected for the predicted high atomic charge states due to significant impact of ion caging and plasma electron screening. This indicates that dense-environment effects can strongly affect local radiation damage-induced structural dynamics.


Assuntos
Proteínas de Bactérias/química , Elétrons , Lasers , Dissulfetos/química , Enxofre/química , Raios X
10.
Artigo em Inglês | MEDLINE | ID: mdl-32224438

RESUMO

A fusion peptide mimicking a part of the sequence of HIV-1 envelope glycoprotein with an additional cysteine at its C-terminus (FP8: AVGIGAVFC) was conjugated to a carrier protein through a linker for development of an HIV-1 vaccine. Since this fusion peptide is very hydrophobic with poor solubility and can self-dimerize via a disulfide bond, co-existence of monomeric and dimeric forms presented a major challenge for residual unconjugated FP8 quantification. A reversed-phase liquid chromatography (RPLC) with UV detection was developed to monitor residual FP8 using an experimental correction factor of 0.85 for UV peak area measurement between FP8 dimer and monomer. Therefore, both forms of unconjugated residual FP8 can be measured based on a single FP8 monomer reference curve. Overall, this study demonstrated that the current purification process can remove free residual FP8 to a low level, <20 µg/mL, which showed negligible impact (<10%) for the conjugated FP8 ratio measurement using another method, amino acid analysis.


Assuntos
Peptídeos/análise , Proteínas Recombinantes de Fusão/análise , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Cisteína/química , Dissulfetos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Conformação Proteica , Multimerização Proteica
11.
Artigo em Inglês | MEDLINE | ID: mdl-32229427

RESUMO

Sclerotinia sclerotiorum is a necrotrophic phytopathogen that has been the subject of several scientific research efforts. Despite the numerous research efforts its proteome remains understudied. This study aimed to identify proteins produced by S. sclerotiorum, thereby increasing the current proteomic knowledge base. Total proteins were extracted from mycelia scraped from five-day old cultures of S. sclerotiorum. The extracted proteins were separated by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and were annotated using the AB Sciex TripleToF 6600 mass spectrometer. Exactly 1471 proteins were reproducibly present in all three replicates. All proteins detected were classified based on their molecular and biological functions. To the knowledge of the authors, this is the most comprehensive proteomic study on S. sclerotiorum (judging by the high number of proteins identified).


Assuntos
Ascomicetos/química , Proteínas Fúngicas/análise , Proteoma/análise , Ascomicetos/metabolismo , Dissulfetos/química , Eletroforese em Gel Bidimensional , Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Micélio/química , Oxirredução , Proteômica
12.
PLoS One ; 15(4): e0230962, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32282813

RESUMO

A designed disulfide-rich ß-hairpin peptide that dimerizes spontaneously served as a hinge-type connection between proteins. Here, we analyze the range of dynamics of this hinge dimer with the aim of proposing new applications for the DNA-encodable peptide and establishing guidelines for the computational analysis of other disulfide hinges. A recent structural analysis based on nuclear magnetic resonance spectroscopy and ion mobility spectrometry revealed an averaged conformation in the hinge region which motivated us to investigate the dynamic behavior using a combination of molecular dynamics simulation, metadynamics and free energy surface analysis to characterize the conformational space available to the hinge. Principal component analysis uncovered two slow modes of the peptide, namely, the opening and closing motion and twisting of the two ß-hairpins assembling the hinge. Applying a collective variable (CV) that mimics the first dominating mode, led to a major expansion of the conformational space. The description of the dynamics could be achieved by analysis of the opening angle and the twisting of the ß-hairpins and, thus, offers a methodology that can also be transferred to other derivatives. It has been demonstrated that the hinge peptide's lowest energy conformation consists of a large opening angle and strong twist but is separated by small energy barriers and can, thus, adopt a closed and untwisted structure. With the aim of proposing further applications for the hinge peptide, we simulated its behavior in the sterically congested environment of a four-helix bundle. Preliminary investigations show that one helix is pushed out and a three-helix bundle forms. The insights gained into the dynamics of the tetra-disulfide peptide and analytical guidelines developed in this study may contribute to the understanding of the structure and function of more complex hinge-type proteins, such as the IgG antibody family.


Assuntos
Peptídeos/química , DNA/química , Dissulfetos/química , Imunoglobulina G/química , Simulação de Dinâmica Molecular , Conformação Proteica
14.
Nat Cell Biol ; 22(4): 476-486, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231310

RESUMO

SLC7A11-mediated cystine uptake is critical for maintaining redox balance and cell survival. Here we show that this comes at a significant cost for cancer cells with high levels of SLC7A11. Actively importing cystine is potentially toxic due to its low solubility, forcing cancer cells with high levels of SLC7A11 (SLC7A11high) to constitutively reduce cystine to the more soluble cysteine. This presents a significant drain on the cellular NADPH pool and renders such cells dependent on the pentose phosphate pathway. Limiting glucose supply to SLC7A11high cancer cells results in marked accumulation of intracellular cystine, redox system collapse and rapid cell death, which can be rescued by treatments that prevent disulfide accumulation. We further show that inhibitors of glucose transporters selectively kill SLC7A11high cancer cells and suppress SLC7A11high tumour growth. Our results identify a coupling between SLC7A11-associated cystine metabolism and the pentose phosphate pathway, and uncover an accompanying metabolic vulnerability for therapeutic targeting in SLC7A11high cancers.


Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Carcinoma de Células Renais/genética , Cistina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Via de Pentose Fosfato/genética , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/secundário , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dissulfetos/metabolismo , Fármacos Gastrointestinais/farmacologia , Glucose/deficiência , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/antagonistas & inibidores , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Pirazóis/farmacologia , Quinolinas/farmacologia , Estresse Fisiológico , Sulfassalazina/farmacologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Exp Parasitol ; 212: 107885, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32234306

RESUMO

A phage-display library was generated using a Bus thalamus scorpion toxin (BTK-2) as a peptide scaffold. BTK-2 belongs to the disulfide-rich family of proteins with pronounced structural stability due to the presence of three disulfide bridges that connects antiparallel beta-sheets and one alpha helix. Using BTK-2 as a phage display scaffold, we introduced mutations in five residues located in the alpha-helix and two residues located in the smaller loop, keeping intact the disulfide bridges to create a peptide phage-displayed library with disulfide-rich family properties. The library was subjected to in vivo and in vitro phage display selections against Trypanosoma evansi, the etiological agent of "Surra", a disease that affects a wide range of mammals. The development of T. evansi specific biomarkers is essential to improve diagnostic methods and epidemiological studies leading to a more accurate clinical decision for the treatment of this disease of economic impact for commercial livestock production. In this study, we identified two disulfide-rich peptides targeting T. evansi parasites. Further specificity studies are necessary to investigate the potential of selected peptides as new biomarkers to aid diagnostic and treatment procedures of T. evansi infections.


Assuntos
Dissulfetos , Peptídeos , Trypanosoma/química , Tripanossomíase/diagnóstico , Tripanossomíase/terapia , Sequência de Aminoácidos , Animais , Biomarcadores , Clonagem Molecular , Dissulfetos/química , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , Oligonucleotídeos/química , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Venenos de Escorpião/química , Venenos de Escorpião/genética
16.
PLoS One ; 15(3): e0230366, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32203539

RESUMO

Posttranslational generation of disulfide bonds catalyzed by bacterial Dsb (disulfide bond) enzymes is essential for the oxidative folding of many proteins. Although we now have a good understanding of the Escherichia coli disulfide bond formation system, there are significant gaps in our knowledge concerning the Dsb systems of other bacteria, including Campylobacter jejuni, a food-borne, zoonotic pathogen. We attempted to gain a more complete understanding of the process by thorough analysis of C8J_1298 functioning in vitro and in vivo. C8J_1298 is a homodimeric thiol-oxidoreductase present in wild type (wt) cells, in both reduced and oxidized forms. The protein was previously described as a homolog of DsbC, and thus potentially should be active in rearrangement of disulfides. Indeed, biochemical studies with purified protein revealed that C8J_1298 shares many properties with EcDsbC. However, its activity in vivo is dependent on the genetic background, namely, the set of other Dsb proteins present in the periplasm that determine the redox conditions. In wt C. jejuni cells, C8J_1298 potentially works as a DsbG involved in the control of the cysteine sulfenylation level and protecting single cysteine residues from oxidation to sulfenic acid. A strain lacking only C8J_1298 is indistinguishable from the wild type strain by several assays recognized as the criteria to determine isomerization or oxidative Dsb pathways. Remarkably, in C. jejuni strain lacking DsbA1, the protein involved in generation of disulfides, C8J_1298 acts as an oxidase, similar to the homodimeric oxidoreductase of Helicobater pylori, HP0231. In E. coli, C8J_1298 acts as a bifunctional protein, also resembling HP0231. These findings are strongly supported by phylogenetic data. We also showed that CjDsbD (C8J_0565) is a C8J_1298 redox partner.


Assuntos
Campylobacter jejuni/enzimologia , Dissulfetos/metabolismo , Proteínas Periplásmicas/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Sequência de Aminoácidos , Campylobacter jejuni/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Oxirredução , Periplasma/enzimologia , Proteínas Periplásmicas/genética , Filogenia , Proteína Dissulfeto Redutase (Glutationa)/genética
17.
Food Chem ; 320: 126665, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32220705

RESUMO

The effects of using proline to solubilise fish myosin under low ionic strength conditions were studied. After solubilising myosin in 0.1 M NaCl containing 5, 10, 15, and 20 mM proline, respectively, it was observed that more than 80% of the myosin was effectively solubilised using 10 mM proline. The addition of 10 mM proline lowered the surface hydrophobicity of myosin from 18.25 to 8.22 mg/g, increased the amount of ß-sheet structure from 33.87% to 46.88%, both of which facilitated solubilisation. As revealed by transfer free energy measurements, the interactions between proline and tyrosine and tryptophan residues were more favourable. Furthermore, the ability of proline to shield hydrophobic sites of myosin and to partially break disulphide bonds helped to form myosin oligomer aggregates. Transmission electron microscopy images verified the effects of proline on myosin proteins. A solubilisation mechanism based mainly on chemical interactions between myosin and proline was proposed.


Assuntos
Proteínas de Peixes da Dieta/química , Miosinas/química , Prolina/química , Tilápia , Animais , Dissulfetos/química , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Concentração Osmolar , Solubilidade , Triptofano , Tirosina
18.
Nat Commun ; 11(1): 1272, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152292

RESUMO

Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/química , Sequência de Aminoácidos , Apoproteínas/química , Dissulfetos/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/ultraestrutura , Humanos , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica , Estabilidade Proteica , Receptores de Glucagon/química
19.
Nat Commun ; 11(1): 1153, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123169

RESUMO

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Endotoxinas/química , Endotoxinas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Dissulfetos/química , Endotoxinas/genética , Endotoxinas/farmacologia , Células HEK293 , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Inseticidas/química , Inseticidas/metabolismo , Inseticidas/farmacologia , Camundongos , Microscopia de Força Atômica , Células NIH 3T3 , Conformação Proteica , Células Sf9
20.
Acta Crystallogr D Struct Biol ; 76(Pt 2): 124-134, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32038043

RESUMO

The members of the CCN (Cyr61/CTGF/Nov) family are a group of matricellular regulatory proteins that are essential to a wide range of functional pathways in cell signalling. Through interacting with extracellular matrix components and growth factors via one of their four domains, the CCN proteins are involved in critical biological processes such as angiogenesis, cell proliferation, bone development, fibrogenesis and tumorigenesis. Here, the crystal structure of the thrombospondin module 1 (TSP1) domain of CCN3 (previously known as Nov) is presented, which shares a similar three-stranded fold with the thrombospondin type 1 repeats of thrombospondin-1 and spondin-1, but with variations in the disulfide connectivity. Moreover, the CCN3 TSP1 domain lacks the typical π-stacked ladder of charged and aromatic residues on one side of the domain that is seen in other TSP1 domains. Using conservation analysis among orthologous domains, it is shown that a charged cluster in the centre of the domain is the most conserved site and this cluster is predicted to be a potential functional epitope for heparan sulfate binding. This variant TSP1 domain has also been used to revise the sequence determinants of TSP1 domains and to derive improved Pfam sequence profiles for the identification of novel TSP1 domains in more than 10 000 proteins across diverse phyla.


Assuntos
Dissulfetos/química , Proteína Sobre-Expressa em Nefroblastoma/química , Trombospondina 1/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Heparitina Sulfato/metabolismo , Humanos , Domínios Proteicos
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