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1.
Biochemistry ; 60(42): 3187-3199, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34613690

RESUMO

α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/efeitos dos fármacos , Quimotripsina/química , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipossomos/metabolismo , Testes de Sensibilidade Microbiana , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Proteólise , Tripsina/química
2.
Arch Insect Biochem Physiol ; 108(3): e21840, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34569086

RESUMO

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), the cotton bollworm, is a destructive pest which is famous for its resistance to a variety of insecticides. RNA interference is a posttranscriptional gene silencing mechanism that has become a popular tool to control insect pests, triggered by double-stranded RNAs (dsRNAs). The effect of ingestion and injection delivery methods of dsRNA related to some protease genes including Trypsin (Ha-TRY39 and Ha-TRY96), Chymotrypsin (Ha-CHY), and Cathepsin L (Ha-CAT) on growth and development of H. armigera was investigated in this study. All protease genes encoded full ORFs and were expressed in all H. armigera larvae stages and tissues. In both injection and feeding bioassays, Ha-RNAi CHY's performance outperformed that of other protease genes. CHY enzyme activity in the midgut of larvae was significantly reduced after treatment with ds-HaCHY. Oral administration of ds-CHY also resulted in significant mortality of H. armigera larvae. However, because of the high RNase activity in the midgut lumen of lepidoptera, a large amount of dsRNA was needed to effectively kill instars of H. armigera. To reduce dsRNA degradation, bacterial expression and dsRNA formulation were used. After oral administration, it was toxic to H. armigera larvae. Before oral administration, bacterial cells were sonicated to increase dsRNA release. The RNA interference efficiency of sonicated bacteria was significantly increased, resulting in higher larval mortality when administered orally. All of these findings point to Ha-CHY as a new candidate for developing an effective dsRNA-based pesticide for H. armigera control.


Assuntos
Mariposas , Peptídeo Hidrolases , RNA de Cadeia Dupla/farmacologia , Animais , Bactérias/genética , Catepsinas/efeitos dos fármacos , Catepsinas/genética , Quimotripsina/efeitos dos fármacos , Quimotripsina/genética , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Mortalidade , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Organismos Geneticamente Modificados , Peptídeo Hidrolases/efeitos dos fármacos , Peptídeo Hidrolases/genética , Controle de Pragas/métodos , Interferência de RNA , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/metabolismo , Tripsina/efeitos dos fármacos , Tripsina/genética
3.
Am J Hum Genet ; 108(10): 1852-1865, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34559995

RESUMO

Genome-wide association studies (GWASs) have discovered 20 risk loci in the human genome where germline variants associate with risk of pancreatic ductal adenocarcinoma (PDAC) in populations of European ancestry. Here, we fine-mapped one such locus on chr16q23.1 (rs72802365, p = 2.51 × 10-17, OR = 1.36, 95% CI = 1.31-1.40) and identified colocalization (PP = 0.87) with aberrant exon 5-7 CTRB2 splicing in pancreatic tissues (pGTEx = 1.40 × 10-69, ßGTEx = 1.99; pLTG = 1.02 × 10-30, ßLTG = 1.99). Imputation of a 584 bp structural variant overlapping exon 6 of CTRB2 into the GWAS datasets resulted in a highly significant association with pancreatic cancer risk (p = 2.83 × 10-16, OR = 1.36, 95% CI = 1.31-1.42), indicating that it may underlie this signal. Exon skipping attributable to the deletion (risk) allele introduces a premature stop codon in exon 7 of CTRB2, yielding a truncated chymotrypsinogen B2 protein that lacks chymotrypsin activity, is poorly secreted, and accumulates intracellularly in the endoplasmic reticulum (ER). We propose that intracellular accumulation of a nonfunctional chymotrypsinogen B2 protein leads to ER stress and pancreatic inflammation, which may explain the increased pancreatic cancer risk in carriers of CTRB2 exon 6 deletion alleles.


Assuntos
Quimotripsina/genética , Neoplasias Pancreáticas/patologia , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Deleção de Sequência , Estudos de Casos e Controles , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/metabolismo
4.
J Fish Dis ; 44(12): 1951-1958, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34363214

RESUMO

The mechanisms enabling fish tapeworms to avoid proteolytic attacks by digestive enzymes of their fish host have been studied in less detail compared with mammalian cestodes. This study aimed to assess the inhibitory ability towards trypsin and chymotrypsin in Eubothrium rugosum, an intestinal parasite of burbot Lota lota, and establish its localization in the tapeworm. To this end, the worms were treated with Triton X-100 followed by differential centrifugation to isolate the tegumental brush border membrane. The protease inhibitory abilities of the worms were mostly determined by their excretory/secretory products released into the incubation medium. These inhibitory abilities proved to be linked mainly with the brush border fractions. Notably, the per cent inhibition of both studied digestive enzymes (trypsin and chymotrypsin) hardly depended on the duration of the parasite exposure in the incubation medium, probably due to intermittent glycocalyx renewal. Improved knowledge on functions of the excretory/secretory proteins produced by fish tapeworms may contribute to a better understanding of host-parasite relations and development of new approaches to the treatment and prevention of diseases caused by pathogenic helminths.


Assuntos
Cestoides/metabolismo , Inibidores de Proteases/metabolismo , Animais , Infecções por Cestoides/enzimologia , Infecções por Cestoides/veterinária , Quimotripsina/antagonistas & inibidores , Doenças dos Peixes/parasitologia , Peixes/parasitologia , Gadiformes , Interações Hospedeiro-Parasita , Inibidores da Tripsina
5.
Front Cell Infect Microbiol ; 11: 668287, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34084756

RESUMO

The oral spirochete Treponema denticola is a keystone periodontal pathogen that, in association with members of a complex polymicrobial oral biofilm, contributes to tissue damage and alveolar bone loss in periodontal disease. Virulence-associated behaviors attributed to T. denticola include disruption of the host cell extracellular matrix, tissue penetration and disruption of host cell membranes accompanied by dysregulation of host immunoregulatory factors. T. denticola dentilisin is associated with several of these behaviors. Dentilisin is an outer membrane-associated complex of acylated subtilisin-family PrtP protease and two other lipoproteins, PrcB and PrcA, that are unique to oral spirochetes. Dentilisin is encoded in a single operon consisting of prcB-prcA-prtP. We employ multiple approaches to study mechanisms of dentilisin assembly and PrtP protease activity. To determine the role of each protein in the protease complex, we have made targeted mutations throughout the protease locus, including polar and nonpolar mutations in each gene (prcB, prcA, prtP) and deletions of specific PrtP domains, including single base mutagenesis of key PrtP residues. These will facilitate distinguishing between host cell responses to dentilisin protease activity and its acyl groups. The boundaries of the divergent promoter region and the relationship between dentilisin and the adjacent iron transport operon are being resolved by incremental deletions in the sequence immediately 5' to the protease locus. Comparison of the predicted three-dimensional structure of PrtP to that of other subtilisin-like proteases shows a unique PrtP C-terminal domain of approximately 250 residues. A survey of global gene expression in the presence or absence of protease gene expression reveals potential links between dentilisin and iron uptake and homeostasis in T. denticola. Understanding the mechanisms of dentilisin transport, assembly and activity of this unique protease complex may lead to more effective prophylactic or therapeutic treatments for periodontal disease.


Assuntos
Quimotripsina , Treponema denticola , Proteínas de Bactérias , Peptídeo Hidrolases
6.
FEBS Lett ; 595(14): 1914-1919, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34080704

RESUMO

Biological structures with highly curved membranes, such as caveolae and transport vesicles, are essential for signal transduction and membrane trafficking. Although membrane proteins in these structures are subjected to physical stress due to the curvature of the lipid bilayers, the effect of this membrane curvature on protein structure and function remains unclear. In this study, we established an experimental procedure to evaluate membrane curvature-induced structural changes in the prototypical potassium channel KcsA. The effect of a large membrane curvature was estimated using fluorescently labeled KcsA by incorporating it into liposomes with a small diameter (< 30 nm). We found that a large membrane curvature significantly affects the activation gate conformation of the KcsA channel.


Assuntos
Proteínas de Bactérias/química , Lipossomos/química , Fosfatidilcolinas/química , Canais de Potássio/química , Potássio/química , Coloração e Rotulagem/métodos , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quimotripsina/química , Corantes Fluorescentes/química , Expressão Gênica , Transporte de Íons , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fosfatidilcolinas/metabolismo , Potássio/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Rodaminas/química , Streptomyces coelicolor/genética
7.
J Mol Biol ; 433(20): 167088, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34087198

RESUMO

I outline how over my career as a protein scientist Machine Learning has impacted my area of science and one of my pastimes, chess, where there are some interesting parallels. In 1968, modelling of three-dimensional structures was initiated based on a known structure as a template, the problem of the pathway of protein folding was posed and bets were taken in the emerging field of Machine Learning on whether computers could outplay humans at chess. Half a century later, Machine Learning has progressed from using computational power combined with human knowledge in solving problems to playing chess without human knowledge being used, where it has produced novel strategies. Protein structures are being solved by Machine Learning based on human-derived knowledge but without templates. There is much promise that programs like AlphaFold based on Machine Learning will be powerful tools for designing entirely novel protein folds and new activities. But, will they produce novel ideas on protein folding pathways and provide new insights into the principles that govern folds?


Assuntos
Aprendizado de Máquina , Dobramento de Proteína , Proteínas/química , Animais , Quimotripsina/química , Humanos , Modelos Moleculares , Conformação Proteica , Software , Tripsina/química
8.
Talanta ; 230: 122341, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33934791

RESUMO

Magnetic titanium dioxide nanoparticles modified with green deep eutectic solvent (DES) composed of choline chloride (ChCl) and xylitol (Xyl) (Fe3O4@TiO2@[ChCl][Xyl]) were synthesized and applied to the solid-phase extraction(MSPE) of chymotrypsin (Chy). The physicochemical properties and morphology of Fe3O4@TiO2@[ChCl][Xyl] was characterized by Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), Zeta potential, X-ray diffraction (XRD), vibrating sample magnetometer (VSM) and transmission electron microscope (TEM). The experiment parameters such as initial concentration of Chy, extraction time, pH value, ionic strength, extraction temperature and sample matrix were effectively optimized. Under the optimal experimental conditions, the extraction capacity of Fe3O4@TiO2@[ChCl][Xyl] obtained a significantly improvement after the modification of Fe3O4@TiO2 nanoparticles by [ChCl][Xyl], and reached up to 347.8 mg g-1. In the elution experiment, 10% sodium dodecyl sulfate-acetic acid (SDS-HAc) was used as eluent, achieving an elution rate of 85.9% for the Chy on Fe3O4@TiO2@[ChCl][Xyl]. And the Fe3O4@TiO2@[ChCl][Xyl] still maintained a good extraction capacity for Chy after six times of reuse. The application result in the extraction of Chy from porcine pancreas crude extract showed a good practical application ability for Chy extraction. All the results indicated that the synthesized Fe3O4@TiO2@[ChCl][Xyl] has good application potential in the extraction of biomolecular molecules such as protein.


Assuntos
Quimotripsina , Nanopartículas , Animais , Fenômenos Magnéticos , Extração em Fase Sólida , Solventes , Espectroscopia de Infravermelho com Transformada de Fourier , Suínos , Titânio
9.
J Chromatogr A ; 1648: 462151, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-33992992

RESUMO

Multi-component adsorption of proteins still requires a better understanding of local phenomena to improve the development of predictive models. In this work, all-atom Molecular Dynamics (MD) simulations were used to investigate the influence of protein charge distribution on the adsorption capacity. The simultaneous adsorption of α-chymotrypsin and lysozyme on a cation exchanger, SP Sepharose FF, was studied through MD simulations and compared to macroscopic isotherm experiments. It appears that the charge distribution is a relevant information to better understand specific phenomena, such as a multilayer adsorption caused by the particular electrostatic profile of α-chymotrypsin. Therefore, MD simulations seem to be an interesting way to visualize and highlight these behaviors.


Assuntos
Cromatografia por Troca Iônica/métodos , Propriedades de Superfície , Adsorção , Quimotripsina/química , Simulação de Dinâmica Molecular , Muramidase/química
10.
Front Immunol ; 12: 634152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054803

RESUMO

Acute hepatopancreatic necrosis disease (AHPND) is a lethal disease in marine shrimp that has caused large-scale mortalities in shrimp aquaculture in Asia and the Americas. The etiologic agent is a pathogenic Vibrio sp. carrying binary toxin genes, pirA and pirB in plasmid DNA. Developing AHPND tolerant shrimp lines is one of the prophylactic approaches to combat this disease. A selected genetic line of Penaeus vannamei was found to be tolerant to AHPND during screening for disease resistance. The mRNA expression of twelve immune and metabolic genes known to be involved in bacterial pathogenesis were measured by quantitative RT-PCR in two populations of shrimp, namely P1 that showed susceptibility to AHPND, and P2 that showed tolerance to AHPND. Among these genes, the mRNA expression of chymotrypsin A (ChyA) and serine protease (SP), genes that are involved in metabolism, and crustin-P (CRSTP) and prophenol oxidase activation system 2 (PPAE2), genes involved in bacterial pathogenesis in shrimp, showed differential expression between the two populations. The differential expression of these genes shed light on the mechanism of tolerance against AHPND and these genes can potentially serve as candidate markers for tolerance/susceptibility to AHPND in P. vannamei. This is the first report of a comparison of the mRNA expression profiles of AHPND tolerant and susceptible lines of P. vannamei.


Assuntos
Perfilação da Expressão Gênica , Hepatopâncreas/metabolismo , Penaeidae/genética , Transcriptoma , Vibrioses/veterinária , Vibrio parahaemolyticus/patogenicidade , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Quimotripsina/genética , Predisposição Genética para Doença , Hepatopâncreas/imunologia , Hepatopâncreas/microbiologia , Hepatopâncreas/patologia , Necrose , Penaeidae/imunologia , Penaeidae/microbiologia , Serina Endopeptidases/genética , Serina Proteases/genética , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/imunologia
11.
Biol Chem ; 402(7): 861-867, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33977684

RESUMO

In order for the intestinal mucosa to absorb dietary proteins they have to be digested into single amino acids or very short peptides of a length of not more than four amino acids. In order to study the efficiency of the digestive endopeptidases to digest folded proteins we have analyzed several target proteins under different conditions, native proteins, heat denatured and acid treated. The three pancreatic serine proteases, trypsin, chymotrypsin, and pancreatic elastase, were found to be remarkable inefficient in cleaving native folded proteins whereas pepsin, which acts at a very low pH (pH 1.2) was much more efficient, possibly due to the denaturing conditions and thereby better accessibility to internal cleavage sites at the low pH. Heat treatment improved the cleavage considerably by all three pancreatic enzymes, but acid treatment followed by return to neutral pH did not have any major effect. Cleavage at the low pH when the protein is in a denatured state, is apparently very efficient. This indicates that pepsin is the prime enzyme cleaving the properly folded native proteins and that the pancreatic enzymes primarily are involved in generating single amino acids or very short peptides for efficient uptake by the intestinal mucosa.


Assuntos
Quimotripsina/química , Elastase Pancreática/química , Pepsina A/química , Tripsina/química , Animais , Bovinos , Quimotripsina/metabolismo , Mucosa Gástrica/enzimologia , Pâncreas/enzimologia , Elastase Pancreática/metabolismo , Pepsina A/metabolismo , Dobramento de Proteína , Suínos , Tripsina/metabolismo
12.
Methods Mol Biol ; 2263: 161-181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877597

RESUMO

Microscale thermophoresis (MST) has become a widely used technique to determine the KD or EC50 of protein-ligand interactions. The method exploits the tendency of macromolecules to migrate along a thermal gradient (i.e., thermophoresis). Differences in thermophoresis as a function of the liganded state of a macromolecule can be measured and assembled into a binding curve that can be analyzed to yield KD. In this protocol, we outline a simple experiment designed for new MST users, with the goal of using readily available, inexpensive materials to plan, execute, and analyze an MST experiment.


Assuntos
Quimotripsina/metabolismo , Soja/metabolismo , Inibidores da Tripsina/metabolismo , Animais , Humanos , Cinética , Proteínas de Plantas/metabolismo , Ligação Proteica , Termodinâmica
13.
Biomed Res Int ; 2021: 6687589, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33855081

RESUMO

The aim of this work is to evaluate the antitumor effect mediated by the proteasome inhibitors of Inula viscosa extracts on skin carcinogenesis. Female Swiss albino mice were divided into five groups depending on the combination of skin cancer-inducing 7,12-dimethylbenz(a)anthracene (DMBA) and extract of Inula viscosa treatments. Histology of the affected skin and measurement of proteasome activity were performed to demonstrate the effect of Inula viscosa on mice. The identification of the molecules responsible for this inhibitory activity was carried out through the docking studies. The results showed that Inula viscosa extracts inhibit the development of papilloma in mice. Therefore, the best chemopreventive action of Inula viscosa was observed on mice in which extract treatment was performed before and after the induction of skin carcinogenesis. It was revealed that the ingestion of extracts Inula viscosa delays the formation of skin papillomas in animals and simultaneously decreases the size and number of papillomas, which is also reflected on the skin histology of the mice treated. Structure-activity relationship information obtained from component of Inula viscosa particularly tomentosin, inuviscolide, and isocosticacid demonstrated that distinct bonding modes in ß 1, ß 2, and ß 5 subunits determine its selectivity and potent inhibition for ß 5 subunit.


Assuntos
Antineoplásicos/uso terapêutico , Inula/química , Extratos Vegetais/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , 9,10-Dimetil-1,2-benzantraceno , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Células CACO-2 , Quimotripsina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos , Simulação de Acoplamento Molecular , Papiloma/tratamento farmacológico , Papiloma/patologia , Extratos Vegetais/análise , Extratos Vegetais/farmacocinética , Extratos Vegetais/farmacologia , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia , Testes de Toxicidade
14.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923636

RESUMO

The crystalline structure of silk fibroin Silk I is generally considered to be a metastable structure; however, there is no definite conclusion under what circumstances this crystalline structure is stable or the crystal form will change. In this study, silk fibroin solution was prepared from B. Mori silkworm cocoons, and a combined method of freeze-crystallization and freeze-drying at different temperatures was used to obtain stable Silk I crystalline material and uncrystallized silk material, respectively. Different concentrations of methanol and ethanol were used to soak the two materials with different time periods to investigate the effect of immersion treatments on the crystalline structure of silk fibroin materials. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman scattering spectroscopy (Raman), Scanning electron microscope (SEM), and Thermogravimetric analysis (TGA) were used to characterize the structure of silk fibroin before and after the treatments. The results showed that, after immersion treatments, uncrystallized silk fibroin material with random coil structure was transformed into Silk II crystal structure, while the silk material with dominated Silk I crystal structure showed good long-term stability without obvious transition to Silk II crystal structure. α-chymotrypsin biodegradation study showed that the crystalline structure of silk fibroin Silk I materials is enzymatically degradable with a much lower rate compared to uncrystallized silk materials. The crystalline structure of Silk I materials demonstrate a good long-term stability, endurance to alcohol sterilization without structural changes, and can be applied to many emerging fields, such as biomedical materials, sustainable materials, and biosensors.


Assuntos
Fibroínas/química , Quimotripsina/metabolismo , Fibroínas/normas , Temperatura Alta , Desnaturação Proteica , Estabilidade Proteica , Proteólise
15.
Int J Nanomedicine ; 16: 2983-2994, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33907402

RESUMO

Background: Bone infections remain one of the most common and serious complications of orthopedic surgery, posing a tremendous economic burden to society and patients. This is because bacteria colonize and multiply on the surface of the implant. The (MMT/PLL)8 multilayer films have been shown to effectively release antibiotics depending on the changes in the microenvironment. Here, vancomycin was loaded into the (MMT/PLL)8 multilayer films, which were prepared to be used as a local delivery system for the treatment of bone infections. Methods: We used the layer-by-layer self-assembly method to prepare VA-loaded coatings (MMT/PLL-VA)8 consisting of montmorillonite (MMT), poly-L-lysine (PLL), and VA. The thickness and surface morphology of coatings were characterized using spectroscopic ellipsometry and scanning electron microscopy (SEM). In order to evaluate the drug release behavior from coatings in different media, we measured the size of the zone of inhibition. Additionally, in vitro antibacterial activity was assessed using the shake-flask culture method and SEM images, while that of in vivo was evaluated by establishing an animal model of bone infection. Results: Our findings revealed that small-molecule antibiotics were successfully loaded into the (MMT/PLL-VA)8 multilayer film structure during the hierarchical self-assembly process and subsequently the multilayer film structure depicted linear growth behavior. The PLL in the multilayer films was progressively degraded which triggered the VA release when contacted with CMS or bacterial infections. The release of VA from multilayer film structure depends on the concentration changes of CMS. Notably, the multilayer films presented great in vitro cell compatibility. Moreover, the prepared antibacterial multilayer films showed excellent antibacterial property by killing more than 99.99% of S. aureus in 24 h. More importantly, we found that multilayer film exhibits good sterilization effect and biocompatibility under the stimulation of bacterial liquid both in vitro and in vivo antibacterial ability tests. Conclusion: Altogether, this study shows that (MMT/PLL-VA)8 multilayer films containing CMS and bacteria-responsive drug release properties posess high bactericidal activity and good biocompatibility. This finding provides a novel strategy for the treatment of bone infections.


Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Materiais Revestidos Biocompatíveis/farmacologia , Implantação Dentária Endo-Óssea/efeitos adversos , Vancomicina/farmacologia , Idoso , Animais , Bentonita/química , Quimotripsina/química , Materiais Revestidos Biocompatíveis/química , Implantes Dentários , Liberação Controlada de Fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Polilisina/química , Infecções Relacionadas à Prótese/tratamento farmacológico , Ratos Sprague-Dawley , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/administração & dosagem , Vancomicina/farmacocinética
16.
J Am Chem Soc ; 143(15): 5709-5716, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33844531

RESUMO

Ultrasensitivity is a ubiquitous emergent property of biochemical reaction networks. The design and construction of synthetic reaction networks exhibiting ultrasensitivity has been challenging, but would greatly expand the potential properties of life-like materials. Herein, we exploit a general and modular strategy to reversibly regulate the activity of enzymes using light and show how ultrasensitivity arises in simple out-of-equilibrium enzymatic systems upon incorporation of reversible photoswitchable inhibitors (PIs). Utilizing a chromophore/warhead strategy, PIs of the protease α-chymotrypsin were synthesized, which led to the discovery of inhibitors with large differences in inhibition constants (Ki) for the different photoisomers. A microfluidic flow setup was used to study enzymatic reactions under out-of-equilibrium conditions by continuous addition and removal of reagents. Upon irradiation of the continuously stirred tank reactor with different light pulse sequences, i.e., varying the pulse duration or frequency of UV and blue light irradiation, reversible switching between photoisomers resulted in ultrasensitive responses in enzymatic activity as well as frequency filtering of input signals. This general and modular strategy enables reversible and tunable control over the kinetic rates of individual enzyme-catalyzed reactions and makes a programmable linkage of enzymes to a wide range of network topologies feasible.


Assuntos
Quimotripsina/metabolismo , Inibidores de Proteases/metabolismo , Biocatálise , Quimotripsina/antagonistas & inibidores , Isomerismo , Cinética , Luz , Microfluídica/métodos , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Especificidade por Substrato , Raios Ultravioleta
17.
Sci Rep ; 11(1): 8648, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33883624

RESUMO

A Bowman-Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman-Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1-10) and temperatures (10-100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains ß sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10-8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (Kd) 2.28 µM.


Assuntos
Sementes/química , Solanum/química , Inibidor da Tripsina de Soja de Bowman-Birk/química , Inibidores da Tripsina/química , Tripsina/química , Sequência de Aminoácidos , Quimotripsina/química , Dicroísmo Circular/métodos , Fabaceae/química , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Temperatura
18.
Biosensors (Basel) ; 11(3)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652946

RESUMO

Chymotrypsin is an important proteolytic enzyme in the human digestive system that cleaves milk proteins through the hydrolysis reaction, making it an interesting subject to study the activity of milk proteases. In this work, we compared detection of chymotrypsin by spectrophotometric dynamic light scattering (DLS) and quartz crystal microbalance (QCM) methods and determined the limit of chymotrypsin detection (LOD), 0.15 ± 0.01 nM for spectrophotometric, 0.67 ± 0.05 nM for DLS and 1.40 ± 0.30 nM for QCM methods, respectively. The sensors are relatively cheap and are able to detect chymotrypsin in 3035 min. While the optical detection methods are simple to implement, the QCM method is more robust for sample preparation, and allows detection of chymotrypsin in non-transparent samples. We give an overview on methods and instruments for detection of chymotrypsin and other milk proteases.


Assuntos
Técnicas Biossensoriais , Quimotripsina/análise , Acústica , Humanos , Técnicas de Microbalança de Cristal de Quartzo
19.
Astrobiology ; 21(4): 405-412, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33784200

RESUMO

The presence of perchlorate ions on Mars raises the question of how these ions influence the biochemistry of any contaminant life introduced into the martian environment, or what selection pressures perchlorate ions exert on any environment that contains these ions, such as the Atacama Desert. In this study, we investigated the structure, stability, and enzyme activity of the model enzyme α-chymotrypsin in the presence of five Mars relevant salts, MgSO4, MgCl2, Mg(ClO4)2, Ca(ClO4)2, and NaClO4. We found that all the perchlorate salts reduced the enzyme activity of α-chymotrypsin in a concentration-dependent manner, with Mg(ClO4)2 and Ca(ClO4)2 having the greatest effect. This observation extends to our structural studies, which show that 1 M Mg(ClO4)2 and Ca(ClO4)2 greatly alter the tertiary structural environment of α-chymotrypsin. We also found that all the perchlorate salts assayed reduced the melting temperature of α-chymotrypsin, whereas the sulfate and chloride salts were able to increase the protein melting temperature. We also demonstrated that a brine containing both perchlorate and sulfate ions exerts the same deleterious effects on α-chymotrypsin's melting temperature and enzyme activity as that of a perchlorate-only brine. This suggests that the perchlorate salts exert a dominant, deleterious effect on protein biochemistry. These results indicate that although perchlorate salts are beneficial to the presence of liquid water due to low eutectic points, they also hamper the habitability of their own environment. Life in such brines would, therefore, have to adapt its cellular machinery to the perchlorate ion's presence or find a way of excluding it from said machinery.


Assuntos
Meio Ambiente Extraterreno , Marte , Quimotripsina , Percloratos , Sais
20.
Nucleic Acids Res ; 49(5): 2721-2739, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33577678

RESUMO

We recently found that toxic PS-ASOs can cause P54nrb and PSF nucleolar mislocalization in an RNase H1-dependent manner. To better understand the underlying mechanisms of these observations, here we utilize different biochemical approaches to demonstrate that PS-ASO binding can alter the conformations of the bound proteins, as illustrated using recombinant RNase H1, P54nrb, PSF proteins and various isolated domains. While, in general, binding of PS-ASOs or ASO/RNA duplexes stabilizes the conformations of these proteins, PS-ASO binding may also cause the unfolding of RNase H1, including both the hybrid binding domain and the catalytic domain. The extent of conformational change correlates with the binding affinity of PS-ASOs to the proteins. Consequently, PS-ASO binding to RNase H1 induces the interaction of RNase H1 with P54nrb or PSF in a 2'-modification and sequence dependent manner, and toxic PS-ASOs tend to induce more interactions than non-toxic PS-ASOs. PS-ASO binding also enhances the interaction between P54nrb and PSF. However, the interaction between RNase H1 and P32 protein can be disrupted upon binding of PS-ASOs. Together, these results suggest that stronger binding of PS-ASOs can cause greater conformational changes of the bound proteins, subsequently affecting protein-protein interactions. These observations thus provide deeper understanding of the molecular basis of PS-ASO-induced protein mislocalization or degradation observed in cells and advance our understanding of why some PS-ASOs are cytotoxic.


Assuntos
Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Fosforotioatos/metabolismo , Ribonuclease H/metabolismo , Linhagem Celular , Quimotripsina , Humanos , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Ligação Proteica , Conformação Proteica , Sinais Direcionadores de Proteínas , RNA/metabolismo , Ribonuclease H/química
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