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1.
Nat Commun ; 11(1): 4817, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968056

RESUMO

Lysozymes are among the best-characterized enzymes, acting upon the cell wall substrate peptidoglycan. Here, examining the invasive bacterial periplasmic predator Bdellovibrio bacteriovorus, we report a diversified lysozyme, DslA, which acts, unusually, upon (GlcNAc-) deacetylated peptidoglycan. B. bacteriovorus are known to deacetylate the peptidoglycan of the prey bacterium, generating an important chemical difference between prey and self walls and implying usage of a putative deacetyl-specific "exit enzyme". DslA performs this role, and ΔDslA strains exhibit a delay in leaving from prey. The structure of DslA reveals a modified lysozyme superfamily fold, with several adaptations. Biochemical assays confirm DslA specificity for deacetylated cell wall, and usage of two glutamate residues for catalysis. Exogenous DslA, added ex vivo, is able to prematurely liberate B. bacteriovorus from prey, part-way through the predatory lifecycle. We define a mechanism for specificity that invokes steric selection, and use the resultant motif to identify wider DslA homologues.


Assuntos
Bdellovibrio bacteriovorus/enzimologia , Bdellovibrio bacteriovorus/metabolismo , Muramidase/química , Muramidase/metabolismo , Periplasma/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bdellovibrio bacteriovorus/genética , Parede Celular , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Muramidase/genética , Mutação , Peptidoglicano/metabolismo , Fenótipo , Conformação Proteica , Especificidade por Substrato
2.
PLoS One ; 15(8): e0237888, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813716

RESUMO

Norovirus, the leading cause of non-bacterial food poisoning, is responsible for several outbreaks associated with bivalves and ready-to-eat food products worldwide. As norovirus is resistant to alcohol, which is commonly used in food manufacturing processes, sodium hypochlorite is used for its inactivation. However, sodium hypochlorite has two disadvantages: it cannot be added to foods, and its effect is significantly reduced in the presence of organic compounds. Thus, a novel disinfectant against norovirus is urgently required for food hygiene. Thermally denatured egg white lysozyme inactivates norovirus; however, the optimal inactivating conditions and the underlying mechanism are unclear. In the present study, the inactivating mechanism of heat-denatured lysozyme against norovirus was analyzed using murine norovirus strain 1 (MNV-1). We found that the inactivating effect was enhanced by adjusting the pH of the lysozyme solution before thermal denaturation to 6.5 or higher. The reaction of heat-denatured lysozyme and MNV-1 was irreversible, and norovirus was completely inactivated after exposure to heat-denatured lysozyme. Furthermore, it was found that lysozyme residues 5-39 contributed to the norovirus-inactivating effect. Notably, the hydrophobicity and positive charges in this region contributed to the norovirus-inactivating effect, as evidenced by the norovirus inactivation test using mutated residues 5-39. These findings are novel and highlight the possible application of heat-denatured lysozyme as a disinfectant against norovirus in a wide range of food processes.


Assuntos
Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Muramidase/metabolismo , Norovirus/fisiologia , Desnaturação Proteica , Inativação de Vírus , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Macrófagos/virologia , Camundongos , Muramidase/química , Peptídeos/química , Domínios Proteicos , Células RAW 264.7
3.
Int J Nanomedicine ; 15: 4705-4716, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636626

RESUMO

Purpose: Ultra-small gold nanoclusters (AuNCs), as emerging fluorescent nanomaterials with excellent biocompatibility, have been widely investigated for in vivo biomedical applications. However, their effects in guiding osteogenic differentiation have not been investigated, which are important for osteoporosis therapy and bone regeneration. Herein, for the first time, lysozyme-protected AuNCs (Lys-AuNCs) are used to stimulate osteogenic differentiation, which have the potential for the treatment of bone disease. Methods: Proliferation of MC3T3E-1 is important for osteogenic differentiation. First, the proliferation rate of MC3T3E-1 was studied by Cell Counting Kit-8 (CCK8) assays. Signaling pathways of PI3K/Akt play central roles in controlling proliferation throughout the body. The expression of PI3K/Akt was investigated in the presence of lysozyme, and lysozyme-protected AuNCs (Lys-AuNCs) by Western blot (WB) and intracellular cell imaging to evacuate the osteogenic differentiation mechanisms. Moreover, the formation of osteoclasts (OC) plays a negative role in the differentiation of osteoblasts. Nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) signaling pathways are used to understand the negative influence of the osteogenic differentiation by the investigation of Raw 264.7 cell line. Raw 264.7 (murine macrophage-like) cells and NIH/3T3 (mouse fibroblast) cells were treated with tyloxapol, and the cell viability was assessed. Raw 264.7 cells have also been used for in vitro studies, on understanding the osteoclast formation and function. The induced osteoclasts were identified by TRAP confocal fluorescence imaging. These key factors in osteoclast formation, such as (NFATc-1, c-Fos, V-ATPase-2 and CTSK), were investigated by Western blot. Results: Based on the above investigation, Lys-AuNCs were found to promote osteogenic differentiation and decrease osteoclast activity. It is noteworthy that the lysozyme (protected template), AuNPs, or the mixture of Lysozyme and AuNPs have negligible effects on osteoblastic differentiation compared to Lys-AuNCs. Conclusion: This study opens up a novel avenue to develop a new gold nanomaterial for promoting osteogenic differentiation. The possibility of using AuNCs as nanomedicines for the treatment of osteoporosis can be expected.


Assuntos
Nanopartículas Metálicas/química , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Ouro/farmacologia , Nanopartículas Metálicas/administração & dosagem , Camundongos , Muramidase/química , Muramidase/metabolismo , Fatores de Transcrição NFATC/metabolismo , Nanomedicina/métodos , Osteoblastos/citologia , Osteoclastos/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7
4.
BMC Bioinformatics ; 21(1): 275, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611389

RESUMO

BACKGROUND: Protein engineering has many applications for industry, such as the development of new drugs, vaccines, treatment therapies, food, and biofuel production. A common way to engineer a protein is to perform mutations in functionally essential residues to optimize their function. However, the discovery of beneficial mutations for proteins is a complex task, with a time-consuming and high cost for experimental validation. Hence, computational approaches have been used to propose new insights for experiments narrowing the search space and reducing the costs. RESULTS: In this study, we developed Proteus (an acronym for Protein Engineering Supporter), a new algorithm for proposing mutation pairs in a target 3D structure. These suggestions are based on contacts observed in other known structures from Protein Data Bank (PDB). Proteus' basic assumption is that if a non-interacting pair of amino acid residues in the target structure is exchanged to an interacting pair, this could enhance protein stability. This trade is only allowed if the main-chain conformation of the residues involved in the contact is conserved. Furthermore, no steric impediment is expected between the proposed mutations and the surrounding protein atoms. To evaluate Proteus, we performed two case studies with proteins of industrial interests. In the first case study, we evaluated if the mutations suggested by Proteus for four protein structures enhance the number of inter-residue contacts. Our results suggest that most mutations proposed by Proteus increase the number of interactions into the protein. In the second case study, we used Proteus to suggest mutations for a lysozyme protein. Then, we compared Proteus' outcomes to mutations with available experimental evidence reported in the ProTherm database. Four mutations, in which our results agree with the experimental data, were found. This could be initial evidence that changes in the side-chain of some residues do not cause disturbances that harm protein structure stability. CONCLUSION: We believe that Proteus could be used combined with other methods to give new insights into the rational development of engineered proteins. Proteus user-friendly web-based tool is available at < http://proteus.dcc.ufmg.br >.


Assuntos
Proteínas/química , Interface Usuário-Computador , Algoritmos , Bases de Dados de Proteínas , Muramidase/química , Muramidase/genética , Muramidase/metabolismo , Mutagênese , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo
5.
Food Chem ; 327: 126920, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32434125

RESUMO

The influence of the timing of inoculation (sequential and simultaneous alcoholic fermentation (AF)/malolactic fermentation (MLF)) on the chemical and sensory properties of red wines was studied. The impact of the encapsulation of Oenococcus oeni into SiO2-alginate hydrogel (Si-ALG) and the addition of lysozyme in wines inoculated with encapsulated bacteria were also analysed. There was a significant influence of the timing of inoculation on the volatile composition of the wines just as on the amino acid and biogenic amine content. The wines produced by simultaneous AF/MLF showed the highest contents of some volatile compounds, such as ethyl esters and terpenes, as well as amino acids and tyramine. Bacterial encapsulation affected the volatile and amino acid profile of the wines, while the biogenic amine composition was not modified. The chemical composition of the wines was not altered by the presence of lysozyme. A trained panel did not perceive substantial differences between treatments.


Assuntos
Aminoácidos/metabolismo , Aminas Biogênicas/metabolismo , Muramidase/metabolismo , Saccharomyces cerevisiae/metabolismo , Dióxido de Silício/química , Vinho/análise , Alginatos/química , Cor , Fermentação , Oenococcus/metabolismo
6.
Ecotoxicol Environ Saf ; 200: 110713, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32464436

RESUMO

Calcutta Leather Complex of the state of West Bengal, India has been designated as an industrially active zone with around 400 active tannery units. This area spanning 4.5 km2 is surrounded by human habitation. The soil of this region is contaminated with metal pollutants and exhibited an alteration in selected physicochemical parameters, namely cation exchange capacity, moisture content, pH, total nitrogen, total organic carbon and water holding capacity. Metaphire posthuma, a common variety of endogeic earthworm inhabiting this region is thus continuously exposed to these toxic metals. Coelomocytes, the chief immune effector cells of earthworm presented a shift in phagocytosis, lysosomal membrane stability, lysozyme and phosphatase activity, physiological apoptosis and cell cycle profile of M. posthuma sampled from the soil of tannery industry. Presence of high concentration of toxic metals and change in the physicochemical characteristics of soil led to a state of cellular stress and immunocompromisation in M. posthuma, a common inhabitant of soil of this region. Experimental endpoints bear ecotoxicological significance as biomarkers of physiological stress in earthworm for monitoring the health of soil around this tannery industrial zone.


Assuntos
Metais/toxicidade , Oligoquetos/efeitos dos fármacos , Poluentes do Solo/toxicidade , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Biomarcadores Ambientais , Humanos , Índia , Indústrias , Lisossomos/efeitos dos fármacos , Muramidase/metabolismo , Oligoquetos/enzimologia , Oligoquetos/imunologia , Oligoquetos/metabolismo , Fagocitose/efeitos dos fármacos , Solo/química
7.
PLoS One ; 15(5): e0232953, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428017

RESUMO

The unpleasant smell released from dead bodies, may serve as an alarm for avoiding certain behaviour or as feeding or oviposition attractants for animals. However, little is known about their effect on the structure and function of proteins. Previously, we reported that using the aroma form of TEMED (a diamine), representative of the "smell of death", could completely inhibit the fibril formation of HEWL, as an antibacterial enzyme, and a model protein for fibrillation studies. To take this further, in this study we investigated the kinetics of TEMED using a number of techniques and in particular X-ray crystallography to identify the binding site(s) of TEMED and search for hotspot(s) necessary to inhibit fibril formation of HEWL. Structural data, coupled with other experimental data reported in this study, revealed that TEMED completely inhibited fibril formation and stabilized the structure of HEWL through enhancement of the CH-Π interaction and binding to an inhibitor hotspot comprised of residues Lys33, Phe34, Glu35 and Asn37 of HEWL. Additionally, results from this study showed that the binding of TEMED increased the activity and thermal stability of HEWL, helping to improve the function of this antibacterial enzyme. In conclusion, the role of the "smell of death", as an important signal molecule affecting the activity and stability of HEWL was greatly highlighted, suggesting that aroma producing small molecules can be signals for structural and functional changes in proteins.


Assuntos
Etilenodiaminas/química , Muramidase/metabolismo , Odorantes/análise , Amiloide/metabolismo , Animais , Antibacterianos , Sítios de Ligação , Galinhas/metabolismo , Cristalografia por Raios X , Feminino , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares
8.
Invest Ophthalmol Vis Sci ; 61(3): 51, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32232350

RESUMO

Purpose: The lysozyme 2 (Lyz2 or LysM) cre mouse is extensively used to achieve genetic manipulation in myeloid cells and it has been widely employed in retinal research. However, LysM has been recently described to be expressed in brain neurons and there is a debate on whether it is also expressed by resident microglia in addition to infiltrating macrophages. Methods: We examined LysM-cre recombination in retinal tissue using a LysM-cre/tdTomato reporter mouse together with immunolabeling for several retinal cell markers. We further compared LysM-cre tdTomato recombination with that of Cdh5-cre driver, which is expressed in both endothelial and hematopoietic cells. Results: LysM-cre was strongly expressed in most microglia/resident macrophages in neonatal retinas (P8) and to a lesser extent in microglia of adult retinas. In addition, there was some neuronal recombination (8 %) of LysM-cre specifically in adult retinal ganglion cells and amacrine cells. After retinal ischemia-reperfusion injury, LysM-cre was strongly expressed in microglia/infiltrating macrophages. Cdh5-cre was expressed in endothelial and myeloid cells of P8 pups retinas. Unexpectedly, Cdh5 showed additional expression in adult mouse retinal ganglion cells and brain neurons. Conclusions: LysM-cre is expressed in macrophages and a subset of microglia together with a small but significant recombination of LysM-cre in the retinal neurons of adult mice. Cdh5 also showed some neuronal expression in both retina and brain of adult mice. These findings should be taken into consideration when interpreting results from central nervous system research using LysM-cre and Cdh5-cre mice.


Assuntos
Antígenos CD/metabolismo , Encéfalo/metabolismo , Caderinas/metabolismo , Integrases/metabolismo , Substâncias Luminescentes/metabolismo , Proteínas Luminescentes/metabolismo , Muramidase/metabolismo , Vasos Retinianos/metabolismo , Animais , Animais Recém-Nascidos , Pesquisa Biomédica , Diagnóstico por Imagem , Endotélio Vascular/metabolismo , Feminino , Genes Reporter , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Neurônios/metabolismo , Recombinação Genética , Traumatismo por Reperfusão/metabolismo , Células Ganglionares da Retina/metabolismo
9.
Transplantation ; 104(9): 1952-1958, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32265415

RESUMO

BACKGROUND: Ischemia-reperfusion injury is inevitable during intestinal transplantation (ITx) and executes a key role in the evolution towards rejection. Paneth cells (PCs) are crucial for epithelial immune defense and highly vulnerable to ischemia-reperfusion injury. We investigated the effect of ITx on PC after reperfusion (T0), during follow-up, and rejection. Moreover, we investigated whether PC loss was associated with impaired graft homeostasis. METHODS: Endoscopic biopsies, collected according to center protocol and at rejection episodes, were retrospectively included (n = 28 ITx, n = 119 biopsies) Biopsies were immunohistochemically co-stained for PC (lysozyme) and apoptosis, and PC/crypt and lysozyme intensity were scored. RESULTS: We observed a decrease in PC/crypt and lysozyme intensity in the first week after ITx (W1) compared with T0. There was a tendency towards a larger decline in PC/crypt (P = 0.08) and lysozyme intensity (P = 0.08) in W1 in patients who later developed rejection compared with patients without rejection. Follow-up biopsies showed that the PC number recovered, whereas lysozyme intensity remained reduced. This persisting innate immune defect may contribute to the well-known vulnerability of the intestine to infection. There was no clear evidence that PCs were affected throughout rejection. CONCLUSIONS: This study revealed a transient fall in PC numbers in the early post-ITx period but a permanent reduction in lysozyme intensity following ITx. Further research is needed to determine the potential clinical impact of PC impairment after ITx.


Assuntos
Rejeição de Enxerto/patologia , Intestinos/transplante , Celulas de Paneth/patologia , Traumatismo por Reperfusão/patologia , Adolescente , Apoptose , Contagem de Células , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Muramidase/metabolismo , Estudos Retrospectivos
10.
Nat Commun ; 11(1): 1231, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144241

RESUMO

We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.


Assuntos
Muramidase/metabolismo , Proteínas Virais/metabolismo , Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Biocatálise , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Simulação de Dinâmica Molecular , Método de Monte Carlo , Muramidase/química , Muramidase/genética , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Virais/química , Proteínas Virais/genética
11.
Chemistry ; 26(24): 5500-5507, 2020 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-32092201

RESUMO

Polydopamine (PD) and melanin species are chemically complex systems, the formation and properties of which are incompletely understood. Inspired by the role of functional amyloids in melanin biosynthesis, this paper examines the influences of the supramolecular structure of amyloids on oxidative polymerization of dopamine. Kinetic analyses on the formation of PD species in the presence of hen egg white lysozyme (HEWL) fibers or soluble HEWL revealed that both forms gave rise to the total quantity of PD species, but the rate of their formation could be accelerated only by the amyloid form. PD species formed with HEWL fibers showed a morphology of bundled fibers, whereas those with soluble HEWL had a mesh-like structure. Amyloid fibers of recombinant Pmel17 had properties similar to those of HEWL fibers in modulating PD formation. The results presented here suggest how nature designs functionality with an amyloid structure and can help understand and engineer chemistries of other functional amyloids.


Assuntos
Amiloide/química , Indóis/química , Melaninas/química , Muramidase/química , Polímeros/química , Amiloide/metabolismo , Animais , Cinética , Muramidase/metabolismo
12.
Chem Asian J ; 15(7): 1025-1029, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32073754

RESUMO

In the life system, the biointerface plays an important role in cell adsorption, platelet adsorption and activation. Therefore, the study of protein adsorption on the biointerface is of great significance for understanding life phenomena and treatment in vitro. In this paper, a chiral biointerface was constructed by the virtue of host-guest interaction between a water-soluble pillar[5]arene (WP5) and phenethylamine (PEA) over a gold surface for adsorption of lysozyme proteins. From the experimental results it was identified that the host-guest biointerface has a high adsorption capacity and strong chiral selectivity. Furthermotre, it was identified that the host-guest interaction plays the decisive role in the enhancement of chirality of the interface, which was much beneficial for increasing protein adsorption and amplifying the capacity of chiral discrimination. Therefore, this work provides a new idea for the construction of biointerface materials with high protein adsorption capacity and high chiral selectivity through supramolecular interaction, which will have potential applications in the fields of biosensors, biocatalysts, biomaterials.


Assuntos
Calixarenos/química , Ouro/química , Fenetilaminas/química , Proteínas/metabolismo , Adsorção , Biocatálise , Materiais Biocompatíveis , Técnicas Biossensoriais , Muramidase/metabolismo , Estereoisomerismo
13.
PLoS One ; 15(1): e0227227, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978114

RESUMO

Many conflicting reports about the involvement of serum amyloid P component (SAP) in amyloid diseases have been presented over the years; SAP is known to be a universal component of amyloid aggregates but it has been suggested that it can both induce and suppress amyloid formation. By using our Drosophila model of systemic lysozyme amyloidosis, SAP has previously been shown to reduce the toxicity induced by the expression of the disease-associated lysozyme variant, F57I, in the Drosophila central nervous system. This study further investigates the involvement of SAP in modulating lysozyme toxicity using histochemistry and spectral analyses on the double transgenic WT and F57I lysozyme flies to probe; i) formation of aggregates, ii) morphological differences of the aggregated lysozyme species formed in the presence or absence of SAP, iii) location of lysozyme and iv) co-localisation of lysozyme and SAP in the fly brain. We found that SAP can counteract the toxicity (measured by the reduction in the median survival time) induced by F57I lysozyme by converting toxic F57I species into less toxic amyloid-like structures, as reflected by the spectral changes that p-FTAA undergoes when bound to lysozyme deposits in F57I-F57I-SAP flies as compared to F57I-F57I flies. Indeed, when SAP was introduced to in vitro lysozyme fibril formation, the endpoint fibrils had enhanced ThT fluorescence intensity as compared to lysozyme fibrils alone. This suggests that a general mechanism for SAP's role in amyloid diseases may be to promote the formation of stable, amyloid-like fibrils, thus decreasing the impact of toxic species formed along the aggregation pathway.


Assuntos
Amiloidose/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Muramidase/metabolismo , Componente Amiloide P Sérico/metabolismo , Amiloide/genética , Amiloide/metabolismo , Amiloide/ultraestrutura , Amiloidose/genética , Amiloidose/patologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Muramidase/genética , Agregados Proteicos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia
14.
Anal Chim Acta ; 1097: 161-168, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-31910956

RESUMO

A new adsorbent based on pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles was developed for specific recognition and efficient adsorption of proteins. Arising from the synergistic effect of specific affinity of apatamer on protein and tunable hydrophobic/hydrophilic property of pH-responsive polymer, the adsorbent exhibited excellent adsorption capacity for target protein. Notably, because of the pH-responsive property of the polymer, the adsorption and desorption process could be regulated through varying environmental pH. The resultant adsorbent that immobilized with lysozyme binding aptamer was successfully applied in specific recognition and efficient adsorption of lysozyme in egg white samples and good recovery results in the range of 95.2-103.2% were obtained. Moreover, the adsorbent immobilized with cytochrome C binding aptamer also exhibited satisfactory adsorption to cytochrome C. The synergistic effect of pH-responsive polymer and aptamer promoted the recognition selectivity and adsorption capacity to target protein, illustrating a facile way for construction of more specific protein adsorbents.


Assuntos
Aptâmeros de Nucleotídeos/química , Nanopartículas de Magnetita/química , Polímeros/química , Adsorção , Conalbumina/análise , Citocromos c/análise , Citocromos c/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Muramidase/análise , Muramidase/metabolismo , Pepsina A/análise , Pepsina A/metabolismo , Albumina Sérica Humana/análise , Tripsina/análise , Tripsina/metabolismo
15.
J Colloid Interface Sci ; 565: 555-566, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31982722

RESUMO

Polyelectrolyte multilayers composed of pharmaceutical grade fucoidan and chitosan have been assembled and studied in terms of their response to physiological solution conditions and the presence of lysozyme. The influence of phosphate buffered saline (PBS) solution on the multilayer was interrogated using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM). The combination of the techniques reveal that the polyelectrolyte multilayers swell when exposed to PBS after build-up and may include a small degree of mass loss as the film swells. The degree of swelling was influenced by the terminating layer of the multilayer. Upon exposure to lysozyme, it was observed that some deswelling occurred, as the enzyme adsorbed onto and permeated into the multilayer. The behaviour of the multilayer as a potential reservoir for lysozyme contrasts with the interaction with bovine serum albumin, which did not penetrate into the multilayer, indicating either exclusion by size or due to the overall net negative charge of the film.


Assuntos
Quitosana/metabolismo , Muramidase/metabolismo , Polieletrólitos/metabolismo , Polissacarídeos/metabolismo , Quitosana/química , Muramidase/química , Tamanho da Partícula , Polieletrólitos/síntese química , Polieletrólitos/química , Polissacarídeos/química , Propriedades de Superfície
16.
Phytochemistry ; 170: 112221, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31790908

RESUMO

The ICChI is a 35-kDa, glycosylated protein isolated from the latex of the weed Ipomoea carnea. It displays chitinase and lysozyme activity, which could be important for the defense against pathogenic fungi, insects and bacteria. The ICChI enzyme was crystallized, and a diffraction data set was collected from a single crystal to 1.42 Å resolution. The crystals belong to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 57.9, c = 172.0 Å, and α = ß = Î³ = 90°. The structure was elucidated by molecular replacement method using a mixed model of three homologous structures from the N-terminal sequence of ICChI. The refined model consists of 272 amino acid residues and has a Rfactor of 18.93% and Rfree of 22.42%. The protein consists of a single globular domain with a (α/ß)8 triosephosphate isomerase barrel fold. Three of the consensus sites for N-glycosylation viz., Asn45, Asn172, and Asn194 containing carbohydrate moieties N-Acetylglucosamine (NAG), mannose, fucose, and xylose. The putative catalytic residues are Asp125, Glu127, and Tyr184. The crystal structure may provide fundamental information of GH18 family chitinases.


Assuntos
Quitinases/metabolismo , Ipomoea/química , Muramidase/metabolismo , Compostos Fitoquímicos/metabolismo , Proteínas de Plantas/metabolismo , Polissacarídeos/metabolismo , Quitinases/química , Ipomoea/metabolismo , Modelos Moleculares , Muramidase/química , Compostos Fitoquímicos/química , Proteínas de Plantas/química , Polissacarídeos/química
17.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140333, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31778816

RESUMO

The integration of enzymes with solid materials is important in many biotechnological applications, including the use of immobilized enzymes for biocatalytic synthesis. The development of functional enzyme-material composites is restrained by the lack of molecular-level insight into the behavior of enzymes in confined, surface-near environments. Here, we review recent advances in surface-sensitive spectroscopic techniques that push boundaries for the determination of enzyme structure and orientation at the solid-liquid interface. We discuss recent evidence from single-molecule studies showing that analyses sensitive to the temporal and spatial heterogeneities in immobilized enzymes can succeed in disentangling the effects of conformational stability and active-site accessibility on activity. Different immobilization methods involve distinct trade-off between these effects, thus emphasizing the need for a holistic (systems) view of immobilized enzymes for the rational development of practical biocatalysts.


Assuntos
Enzimas Imobilizadas/metabolismo , Biocatálise , Domínio Catalítico , Enzimas Imobilizadas/química , Muramidase/química , Muramidase/metabolismo , Conformação Proteica , Desdobramento de Proteína , Dióxido de Silício/química
18.
Biosens Bioelectron ; 148: 111816, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31678823

RESUMO

Protein fibrous aggregation is associated with many neurodegenerative diseases including Alzheimer's and Parkinson's diseases. To modulate the process, a number of fibrillation inhibitors have been reported, although their working mechanism remains vague, calling for new means to decipher their interaction. Herein, we identified and characterized a novel inhibitor called Crocein Orange G (COG), which inhibited the nucleation and impeded the protofibril formation, revealed by various experimental approaches as well as molecular docking. In particular, the surface-enhanced Raman spectroscopy (SERS) helps to identify the binding sites and illustrate the interaction mechanism and fibrillation process by using Ag IMNPs as SERS substrate for a label-free detection. Combining with molecular docking, the SERS-based approach provides structural information concerning protein-ligand interaction and protein fibrillation. This study suggests that SERS can be a powerful new means to study the interaction between inhibitors and amyloid proteins and can potentially be a common tool for amyloid research. Strikingly, the SERS signal of COG corresponds very well with the state of protein fibrillation, hinting its function as an amyloid SERS signal amplifier. Therefore, this study provides a new means to monitor and interfere amyloid fibrillation.


Assuntos
Amiloide/metabolismo , Compostos Azo/farmacologia , Naftalenossulfonatos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Análise Espectral Raman/métodos , Amiloide/química , Técnicas Biossensoriais/métodos , Humanos , Insulina/química , Insulina/metabolismo , Simulação de Acoplamento Molecular , Muramidase/química , Muramidase/metabolismo , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , Prata/química
19.
Colloids Surf B Biointerfaces ; 185: 110589, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31707228

RESUMO

Built upon our interest in illustrating the complexity of protein adsorption onto chromatographic supports and to understand the rule of nonspecific interactions in the ion exchange adsorption process, a traditional model system (lysozyme - carboxymethyl cellulose) was used to determine the charge influence during biomolecule adsorption. Flow microcalorimetry (FMC) was exploit as a dynamic technique that provides adsorption and desorption heat signals for a specific system, permitting an improved understanding of the driving forces and mechanisms involved during the interaction. For this purpose, measurements were made at pH 8 at both absence and presence of salt (NaCl 50 mM) and compared with previous studies performed at pH 5. Distinct FMC profiles were observed regarding pH. For most of the experiments, two exothermic heat signals are observed at pH 8 while at pH 5 one endothermic and one exothermic peak are shown. This difference was justified with a less energy demanding for desolvation at pH 8. Lysozyme adsorption was shown to be a multi-step process involving desolvation, primary protein adsorption and secondary adsorption after reorientation with distinct contributions to the overall energy. At pH 8, the exothermic contribution to the adsorption process is lower compared to pH 5, which is justified by the lower charge density that lysozyme presents at pH 8 compared to pH 5.


Assuntos
Calorimetria/métodos , Carboximetilcelulose Sódica/metabolismo , Cátions/química , Cromatografia por Troca Iônica/métodos , Muramidase/metabolismo , Cloreto de Sódio/química , Adsorção , Carboximetilcelulose Sódica/química , Humanos , Concentração de Íons de Hidrogênio , Muramidase/química , Muramidase/isolamento & purificação , Termodinâmica
20.
Biophys Chem ; 254: 106265, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31669866

RESUMO

The impact of the differently hydrated non-electrolytes (protein structure destabilizers) on the fibrillation of hen egg white lysozyme (HEWL) was investigated. Two isomeric urea derivatives i.e. butylurea (BU) and N,N,N',N'-tetramethylurea (TMU) were chosen as a tested compounds. The obtained results show that butylurea exerts greater impact on HEWL and its fibrillation than tetramethylurea. Both substances decrease the time of induction of the fibrillation (lag time) but only BU increases the efficiency of amyloidogenesis. For the systems with equivalent reduction of the HEWL stability (250mM BU and 500mM TMU) the not-equivalent increase of the protein fibrillation was recorded (higher for BU). This fact suggests that specific interactions with protein, possibly water mediated, are responsible for the action of the tested substances.


Assuntos
Amiloide/química , Muramidase/química , Água/química , Animais , Galinhas , Dicroísmo Circular , Microscopia de Força Atômica , Muramidase/metabolismo , Estabilidade Proteica , Soluções/química , Ureia/química
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