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1.
Chem Pharm Bull (Tokyo) ; 67(9): 897-903, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474726

RESUMO

The word "theranostics," a portmanteau word made by combining "therapeutics" and "diagnostics," refers to a personalized medicine concept. Recently, the word, "radiotheranostics," has also been used in nuclear medicine as a term that refer to the use of radioisotopes for combined imaging and therapy. For radiotheranostics, a diagnostic probe and a corresponding therapeutic probe can be prepared by introducing diagnostic and therapeutic radioisotopes into the same precursor. These diagnostic and therapeutic probes can be designed to show equivalent pharmacokinetics, which is important for radiotheranostics. As imaging can predict the absorbed radiation dose and thus the therapeutic and side effects, radiotheranostics can help achieve the goal of personalized medicine. In this review, I discuss the use of radiolabeled probes targeting bone metastases, sigma-1 receptor, and αVß3 integrin for radiotheranostics.


Assuntos
Neoplasias Ósseas/diagnóstico , Compostos Radiofarmacêuticos/química , Animais , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/radioterapia , Meios de Contraste/química , Meios de Contraste/metabolismo , Humanos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/uso terapêutico , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Receptores sigma/química , Receptores sigma/metabolismo , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Distribuição Tecidual
2.
J Microbiol Biotechnol ; 29(8): 1310-1315, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370115

RESUMO

Hyaluronidases enhance therapeutic drug transport by breaking down the hyaluronan barrier to lymphatic and capillary vessels, facilitating their tissue absorption. Commercially available hyaluronidases are bovine in origin; however, they pose risks such as bovine spongiform encephalopathy. The present study aimed to develop a novel, highly active hyaluronidase and assess its function. Therefore, in order to find the most efficient active hyaluronidase, we produced several shortened hyaluronidases with partial removal of the N- or C-terminal regions. Moreover, we created an enzyme that connected six histidines onto the end of the hyaluronidase C-terminus. This simplified subsequent purification using Ni2+ affinity chromatography, making it feasible to industrialize this highly active recombinant hyaluronidase which exhibited catalytic activity equal to that of the commercial enzyme. Therefore, this simple and effective isolation method could increase the availability of recombinant hyaluronidase for research and clinical purposes.


Assuntos
Histidina/metabolismo , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Oligopeptídeos/metabolismo , Proteínas Recombinantes , Animais , Bovinos , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Células HEK293 , Humanos , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/isolamento & purificação , Concentração de Íons de Hidrogênio , Temperatura Ambiente
3.
Food Chem ; 297: 124931, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253328

RESUMO

Enzyme hydrolysis of corn gluten meal (CGM) is a promising process to prepare oligopeptides with high Fischer ratios (HFOPs). However, the relationship between Fischer ratios and enzyme hydrolysis approaches remains poorly understood. In this study, peptidomes of varying corn protein hydrolysates (CPHs) before and after activated carbon adsorption were profiled and analyzed according to sequence composition and chain length. Fischer ratios of HFOPs depended on sequences in CPHs by differing enzyme hydrolysis approaches, especially branched-chain amino acid (BCAA)-aromatic amino acid (AAA)-containing-oligopeptides. Activated carbon adsorption increased BCAA-containing-oligopeptide contents and decreased oligopeptide contents including AAAs, preferring BCAA-AAA-containing-oligopeptides with long chain length. Employing a three-enzyme hydrolysis approach, HFOPs were obtained with a yield of 49%, comprising 90% of dipeptides and tripeptides and possessing additional bioactivities. This work revealed the mechanism of HFOP production depending on the release and selective removal of oligopeptides and confirmed CGM was a promising alternative for value-added HFOP production.


Assuntos
Glutens/metabolismo , Oligopeptídeos/química , Zea mays/metabolismo , Adsorção , Aminoácidos Aromáticos/química , Aminoácidos de Cadeia Ramificada/química , Carbono/química , Cromatografia Líquida de Alta Pressão , Hidrólise , Peso Molecular , Oligopeptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Espectrometria de Massas em Tandem
4.
Cell Physiol Biochem ; 53(1): 87-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31204440

RESUMO

BACKGROUND/AIMS: Different components of the tumor microenvironment can be either tumor-promoting or tumor-suppressive agents depending on factors which are not fully understood. Fibulins are components of the extracellular matrix from different tissues and constitute a clear example of this dual function. In fact, fibulins may either support tumor growth or abolish progression of malignant cells depending on the crosstalk between tumor cells and their surrounding stroma through mechanisms that remain to be elucidated. Among all fibulins, fibulin-5 contains a particular structural hallmark which consists in the presence of a RGD motif within its architecture. Previous reports have highlighted the importance of the interaction of this motif with integrins, and not only in normal functions but also in a tumor context. METHODS: Site-Directed Mutagenesis technique was employed to introduce the change RGD to RGE (RGD-to-RGE) within Fbln5 cDNA sequence. Cell proliferation was measured using the MTT assay or by counting Ki-67 positive cell nuclei. Cell adhesion was analysed using culture plates coated with different extracellular matrix components. Cell invasion was evaluated using 24-well Matrigel-coated invasion chambers, and mammosphere formation was monitored using ultralow attachment culture plates. BALB/c mice were employed to induce subcutaneous tumors. RESULTS: The RGD-to-RGE change alters the capacity of breast cancer cells to adhere to different extracellular matrix proteins as well as to αvß3 and α5ß1 integrins, and promotes protumor effects using different cell-based assays. Moreover, 4T1 cells, a mouse breast cancer cell line model, shows an increased capacity to generate tumors when exogenously expresses fibulin-5 with a RGD-to-RGE change, and such capacity is similar to that shown for 4T1 cells with an interfered Fbln5 gene. CONCLUSION: These data highlight the importance of the RGD motif of fibulin-5 to induce antitumor effects and provide new insights into the involvement of fibulins in tumor processes.


Assuntos
Adesão Celular/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Oligopeptídeos/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/uso terapêutico , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Oligopeptídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transplante Homólogo , Vimentina/metabolismo
5.
Eur J Med Chem ; 176: 105-116, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31100648

RESUMO

Development of peptide-based conjugates for targeted tumour therapy is a current research topic providing new possibilities in cancer treatment. In this study, VHLGYAT heptapeptide selected by phage display technique for HT-29 human colon cancer was investigated as homing peptide for drug delivery. Daunomycin was conjugated to the N-terminus of the peptide directly or through Cathepsin B cleavable spacers. Conjugates showed moderate in vitro cytostatic effect. Therefore, sequence modifications were performed by Ala-scan and positional scanning resulting in conjugates with much higher bioactivity. Conjugates in which Gly was replaced by amino acids with bulky apolaric side chains provided the best efficacy. The influence of the cellular uptake, stability and drug release on the anti-tumour activity was investigated. It was found that mainly the difference in the cellular uptake of the conjugates generated the distinct effect on cell viability. One of the most efficient conjugate Dau = Aoa-LRRY-VHLFYAT-NH2 showed tumour growth inhibition on orthotopically developed HT-29 colon cancer in mice with negligible toxic side effect compared to the free drug. We also indicate that this sequence is not specific to HT-29 cells, but it has a remarkable effect on many other cancer cells. Nevertheless, the Phe-containing conjugate was more active in all cases compared to the conjugate with the parent sequence. The literature data suggested that this sequence is highly overlapped with peptides that recognize Hsp70 membrane bound protein overexpressed in many types of tumours.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Daunorrubicina/análogos & derivados , Daunorrubicina/uso terapêutico , Oligopeptídeos/uso terapêutico , Pró-Fármacos/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Catepsina B/metabolismo , Proliferação de Células/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular/métodos , Daunorrubicina/metabolismo , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Feminino , Células HT29 , Humanos , Camundongos SCID , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/metabolismo , Proteólise , Ratos , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Chemistry ; 25(41): 9728-9736, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31062438

RESUMO

Enzyme-mediated catalysis is attributed to enzyme-substrate interactions, with models such as "induced fit" and "conformational selection" emphasizing the role of protein conformational transitions. The dynamic nature of the protein structure, thus, plays a crucial role in molecular recognition and substrate binding. As large-scale protein motions are coupled to water motions, hydration dynamics play a key role in protein dynamics, and hence, in enzyme catalysis. Here, microfluidic techniques and time-dependent fluorescence Stokes shift (TDFSS) measurements are employed to elucidate the role of nanoscopic water dynamics in the interaction of an enzyme, α-Chymotrypsin (CHT), with a substrate, Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) in the cationic reverse micelles of benzylhexadecyldimethylammonium chloride (BHDC/benzene) and anionic reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT/benzene). The kinetic pathways unraveled from the microfluidic setup are consistent with the "conformational selection" fit for the interaction of CHT with AMC in the cationic reverse micelles, whereas an "induced fit" mechanism is indicated for the anionic reverse micelles. In the cationic reverse micelles of BHDC, faster hydration dynamics (≈550 ps) aid the pathway of "conformational selection", whereas in the anionic reverse micelles of AOT, the significantly slower dynamics of hydration (≈1600 ps) facilitate an "induced fit" mechanism for the formation of the final enzyme-substrate complex. The role of water dynamics in dictating the mechanism of enzyme-substrate interaction becomes further manifest in the neutral reverse micelles of Brij-30 and Triton X-100. In the former, the faster water dynamics aid the "conformational selection" pathway, whereas the significantly slower dynamics of water molecules in the latter are conducive to the "induced fit" mechanism in the enzyme-substrate interaction. Thus, nanoscopic water dynamics act as a switch in modulating the pathway of recognition of an enzyme (CHT) by the substrate (AMC) in reverse micelles.


Assuntos
Quimotripsina/metabolismo , Cumarínicos/metabolismo , Dispositivos Lab-On-A-Chip , Micelas , Oligopeptídeos/metabolismo , Água/metabolismo , Ânions/metabolismo , Cátions/metabolismo , Desenho de Equipamento , Fluorescência , Cinética , Especificidade por Substrato , Tensoativos/metabolismo
7.
Analyst ; 144(12): 3773-3781, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31089613

RESUMO

MDM2 is a well-known oncoprotein overexpressed in a variety of cancers, and the identification of inhibitors that disrupt the MDM2/p53 interaction is of great interest in anticancer drug development. Here we designed a platform for the facile and visualizable identification of inhibitors of MDM2 using co-expressed protein complexes of MDM2/p53. A hexahistidine-tag on MDM2 allows the binding of the protein complex to the Ni-NTA affinity resin, while the fluorescent protein fused to p53 enables the direct visualization of the interaction of p53 with MDM2. Hence, the inhibition of the MDM2/p53 interaction can be observed with the naked eye. The assay can be set up by directly loading cell lysate to the Ni-NTA affinity resin, and no chemical modification of proteins is needed. In addition to the qualitative analyses, the binding affinity of inhibitors to the MDM2 protein can be quantified by fluorescence titration. The applications of this system have been verified using small molecules and peptide inhibitors. As a proof of concept, we screened a small library using this platform. Interestingly, two types of novel inhibitors of MDM2, including cyclohexyl-triphenylamine derivatives and platinum complexes, were identified and their binding affinities were obtained. Quantitative measurements show that these new types of inhibitors demonstrate a high binding affinity (up to Kd = 51.9 nM) to MDM2.


Assuntos
Bioensaio/métodos , Proteínas Luminescentes/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Compostos de Anilina/química , Cromatografia de Afinidade/métodos , Complexos de Coordenação/química , Escherichia coli/genética , Histidina/genética , Histidina/metabolismo , Humanos , Medições Luminescentes/métodos , Proteínas Luminescentes/genética , Simulação de Acoplamento Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptídeos/química , Platina/química , Estudo de Prova de Conceito , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética
8.
Nanoscale ; 11(17): 8210-8218, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30973561

RESUMO

Water in nanoscale-confined geometries has unique physicochemical properties in contrast to bulk water, and is believed to play important roles in biological processes although there is less direct information available in the literature. Here, we report the self-assembly behaviors of a neurodegenerative disease related peptide termed GAV-9 encapsulated in mica-graphene nanocapillaries interacting with water nanofilms condensed under ambient conditions, based on atomic force microscopy (AFM) imaging and molecular dynamics (MD) simulations. The results revealed that, upon increase in the humidity, the GAV-9 peptide monomers adsorbed the confined water molecules and transitioned to unexpected hydrogel-like structures. Our MD simulations also suggested that in the confined mica-graphene nanocapillaries, the GAV-9 peptide monomers would indeed form water-rich hydrogel structures instead of highly ordered nanofilaments. The interfacial water confined in the mica-graphene nanocapillary is found to be crucial for such a transition. Moreover, the distribution of confined water layers largely depended on the locations of the preformed peptide nanofilaments, and the peptide nanofilaments further assembled into nanosheets with the water layer increasing, but depolymerized to amorphous peptide assemblies with the water layer decreasing. The polymerization and depolymerization of the peptide nanofilaments could be controlled in a reversible manner. Our results have supplied a simplified model system to uncover the effects of the confined interfacial water on the biological process at the molecular level.


Assuntos
Silicatos de Alumínio/química , Grafite/química , Nanoestruturas/química , Oligopeptídeos/química , Água/química , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo
9.
Mar Drugs ; 17(4)2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30935056

RESUMO

A protein extract was generated from the macroalga Ulva lactuca, which was subsequently hydrolysed using the food-grade enzyme papain and angiotensin-converting Enzyme I and renin inhibitory peptides identified using a combination of enrichment strategies employing molecular weight cutoff filtration and mass spectrometry analysis. The generated hydrolysates with the most promising in vitro activity were further purified using preparative RP-HPLC and characterised. The 1 kDa hydrolysate (1 kDa-UFH), purified and collected by preparative RP-HPLC at minutes 41‒44 (Fr41‒44), displayed statistically higher ACE-I inhibitory activities ranging from 96.91% to 98.06%. A total of 48 novel peptides were identified from these four fractions by LC-MS/MS. A simulated gastrointestinal digestion of the identified peptide sequences was carried out using in silico enzyme cleavage simulation tools, resulting in 86 peptide sequences that were further assessed for their potential activity, toxicity and allergenicity using multiple predictive approaches. All the peptides obtained in this study were predicted to be non-toxic. However, 28 out of the 86 novel peptides released after the in silico gastrointestinal digestion were identified as potential allergens. The potential allergenicity of these peptides should be further explored to comply with the current labelling regulations in formulated food products containing U. lactuca protein hydrolysates.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Hidrolisados de Proteína/farmacologia , Ulva/metabolismo , Alérgenos/farmacologia , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Anti-Hipertensivos/farmacologia , Simulação por Computador , Humanos , Hidrólise , Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Hidrolisados de Proteína/química , Hidrolisados de Proteína/isolamento & purificação , Alga Marinha/química , Ulva/química , Ulva/citologia
10.
Nutrients ; 11(4)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987324

RESUMO

Alcalase- generated potato protein hydrolysate (APPH) is a potential bioactive peptide against diabetes mellitus (DM) and DM-associated secondary effects in animal models. The aim of the present study was to find the efficiency of a deca-peptide DIKTNKPVIF (DF) from APPH against DM. Six-week-old male ICR mice were divided into the following groups: Control, Control+DF (received 50 mg/kg DF), streptozotocin (STZ)-induced DM group, DM+Acarbose group (20 mg/kg of acarbose), DM+DF-L (25 mg/kg of DF), DM+DF-H (50 mg/kg of DF), and DM+APPH (50 mg/kg of APPH). Comparable to APPH, treatment with DF effectively regulated blood glucose level and also controlled plasma total glycerol (TG), total cholesterol (TC), insulin, and HbA1c levels in DM animals. DF treatment also showed evidence of ameliorating DM-associated damages in the pancreatic islets and in the liver, heart, and kidney tissues. Therefore, the results demonstrate that the short synthetic peptide-DF may effectively provide protection against DM-associated damages.


Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/prevenção & controle , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Oligopeptídeos/farmacologia , Proteínas de Plantas/metabolismo , Hidrolisados de Proteína/metabolismo , Solanum tuberosum/metabolismo , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Hemoglobina A Glicada/metabolismo , Hipoglicemiantes/metabolismo , Insulina/sangue , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Lipídeos/sangue , Masculino , Camundongos Endogâmicos ICR , Oligopeptídeos/metabolismo , Estreptozocina , Subtilisinas/metabolismo
11.
Eur J Med Chem ; 174: 159-180, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31035238

RESUMO

Inhibitors and nucleic acid based techniques were two main approaches to interfere with protein signaling and respective cascade in the past. Until recently, a new class of small molecules named proteolysis-targeting chimeras (PROTACs) have emerged. Each contains a target warhead, a linker and an E3 ligand. These bifunctional molecules recruit E3 ligases and target specific proteins for degradation via the ubiquitin (Ub) proteasome system (UPS). The degradation provides several advantages over inhibition in potency, selectivity and drug resistance. Thus, a variety of small molecule PROTACs have been discovered so far. In this review, we summarize the biological mechanism, advantages and recent progress of PROTACs, trying to offer an outlook in development of drugs targeting degradation in future.


Assuntos
Oligopeptídeos/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Ligantes , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas/química , Proteólise/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
12.
Science ; 363(6433)2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30898901

RESUMO

Physical damage to cells leads to the release of immunomodulatory peptides to elicit a wound defense response in the surrounding tissue. In Arabidopsis thaliana, the plant elicitor peptide 1 (Pep1) is processed from its protein precursor, PRECURSOR OF PEP1 (PROPEP1). We demonstrate that upon damage, both at the tissue and single-cell levels, the cysteine protease METACASPASE4 (MC4) is instantly and spatiotemporally activated by binding high levels of Ca2+ and is necessary and sufficient for Pep1 maturation. Cytosol-localized PROPEP1 and MC4 react only after loss of plasma membrane integrity and prolonged extracellular Ca2+ entry. Our results reveal that a robust mechanism consisting of conserved molecular components links the intracellular and Ca2+-dependent activation of a specific cysteine protease with the maturation of damage-induced wound defense signals.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Cálcio/metabolismo , Cisteína Endopeptidases/metabolismo , Imunomodulação , Imunidade Vegetal , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Citosol/enzimologia , Oligopeptídeos/metabolismo
13.
Int J Mol Sci ; 20(6)2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901841

RESUMO

Synergizing integrin and cell-membrane heparan sulfate proteoglycan signaling on biomaterials through peptidic sequences is known to have beneficial effects in the attachment and behavior of osteoblasts; however, controlling the exact amount and ratio of peptides tethered on a surface is challenging. Here, we present a dual molecular-based biointerface combining integrin (RGD) and heparin (KRSR)-binding peptides in a chemically controlled fashion. To this end, a tailor-made synthetic platform (PLATF) was designed and synthesized by solid-phase methodologies. The PLATF and the control linear peptides (RGD or KRSR) were covalently bound to titanium via silanization. Physicochemical characterization by means of contact angle, Raman spectroscopy and XPS proved the successful and stable grafting of the molecules. The biological potential of the biointerfaces was measured with osteoblastic (Saos-2) cells both at short and long incubation periods. Biomolecule grafting (either the PLATF, RGD or KRSR) statistically improved (p < 0.05) cell attachment, spreading, proliferation and mineralization, compared to control titanium. Moreover, the molecular PLATF biointerface synergistically enhanced mineralization (p < 0.05) of Saos-2 cells compared to RGD or KRSR alone. These results indicate that dual-function coatings may serve to improve the bioactivity of medical implants by mimicking synergistic receptor binding.


Assuntos
Membrana Celular/metabolismo , Integrinas/metabolismo , Oligopeptídeos/metabolismo , Osteoblastos/metabolismo , Proteoglicanas/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Fenômenos Químicos , Materiais Revestidos Biocompatíveis/química , Matriz Extracelular/metabolismo , Integrinas/química , Oligopeptídeos/química , Proteoglicanas/química , Análise Espectral
14.
Protein Pept Lett ; 26(5): 348-356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816077

RESUMO

BACKGROUND: The Bursa of Fabricius is an acknowledged central humoral immune organ unique to birds, which provides an ideal research model on the immature B cell development. OBJECTIVE: In this article, our motivation is to study the role on sIgM and establish the molecular basis and functional processes of Bursal Hexapeptide (BHP) in avian immature B cells DT40 cell lines. METHODS: In this article, we detected the expressions of sIgM mRNA with qPCR in DT40 cells with BHP treatment, and investigated the gene expression profiles of BHP-treated DT40 cells, employing microarray analyses. Also, to validate the differentially expressed genes, we performed KEGG pathway and Gene Ontology analysis in the BHP-treated DT40 cells. Finally, we comparatively analyzed the similar regulated genes and their involved immune functional processes between DT40 cell and mouse immature B cell line WEHI231 cell with BHP treatment. RESULTS: Following the proposed framework, we proved that the BHP enhanced the mRNA expression levels of IgM in DT40 cells, and induced 460 upregulated genes and 460 downregulated genes in BHP-treated DT40 cells. The pathway analysis showed that the differentially regulated genes in DT40 cell line with BHP treatment were involved in 12 enrichment pathways, in which Toll-like receptor signaling pathway was the vital pathways, and cytokine-cytokine receptor interaction and Jak-STAT signaling pathway were another two important pathways in BHP-treated DT40 cells. Moreover, BHP induced the immune related biological processes in BHP-treated DT40 cells, including T cell related, cytokine related, lymphocyte related, and innate immune response GO terms. Finally, the comparatively analysis showed that there were two downregulated genes GATA3 and IFNG to be found co-existed among the differentially expressed genes in BHP-treated DT40 cell and WEHI231 cells, which shared some same immune related functional processes in both cell lines. CONCLUSION: After the applying the framework, we proved the inducing roles and the gene expression profiles of BHP on avian immature B cells, and verified some molecular basis from the KEGG and GO analysis. These results provided the insight for mechanism on immature B cell differentiation, and offer the essential direction for the vaccine improvement.


Assuntos
Oligopeptídeos/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Animais , Bolsa de Fabricius , Linhagem Celular , Galinhas , Imunidade Inata , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Camundongos , Oligopeptídeos/farmacologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/imunologia , RNA Mensageiro/metabolismo , Transdução de Sinais , Especificidade da Espécie , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo
15.
Med Mycol ; 57(Supplement_2): S228-S232, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30816973

RESUMO

In airways of immunocompromised patients and individuals with cystic fibrosis, Pseudomonas aeruginosa and Aspergillus fumigatus are the most common opportunistic bacterial and fungal pathogens. Both pathogens form biofilms and cause acute and chronic illnesses. Previous studies revealed that P. aeruginosa is able to inhibit A. fumigatus biofilms in vitro. While numerous P. aeruginosa molecules have been shown to affect A. fumigatus, there never has been a systematic approach to define the principal causative agent. We studied 24 P. aeruginosa mutants, with deletions in genes important for virulence, iron acquisition, or quorum sensing, for their ability to interfere with A. fumigatus biofilms. Cells, planktonic or biofilm culture filtrates of four P. aeruginosa mutants, pvdD-pchE-, pvdD-, lasR-rhlR-, and lasR-, inhibited A. fumigatus biofilm metabolism or planktonic A. fumigatus growth significantly less than P. aeruginosa wild type. The common defect of these four mutants was a lack in the production of the P. aeruginosa siderophore pyoverdine. Pure pyoverdine affected A. fumigatus biofilm metabolism, and restored inhibition by the above mutants. In lungs from cystic fibrosis patients, pyoverdine production and antifungal activity correlated. The key inhibitory mechanism for pyoverdine was iron-chelation and denial of iron to A. fumigatus. Further experiments revealed a counteracting, self-protective mechanism by A. fumigatus, based on A. fumigatus siderophore production.


Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/crescimento & desenvolvimento , Interações Microbianas , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Infecções Respiratórias/microbiologia , Aspergilose/patologia , Humanos , Mutação , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética , Infecções Respiratórias/patologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
16.
Mol Cell ; 73(5): 1075-1082.e4, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849388

RESUMO

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.


Assuntos
Anticorpos/metabolismo , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligopeptídeos/metabolismo , Análise Serial de Proteínas/métodos , Afinidade de Anticorpos , Especificidade de Anticorpos , Automação Laboratorial , Sítios de Ligação de Anticorpos , Catálise , Análise Mutacional de DNA/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Cinética , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Oligopeptídeos/genética , Análise Serial de Proteínas/instrumentação , Ligação Proteica , Engenharia de Proteínas , Fluxo de Trabalho
17.
Mol Pharmacol ; 95(5): 519-527, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30808671

RESUMO

Muscle ischemia, associated with peripheral artery disease (PAD), leads to the release of proinflammatory mediators that decrease extracellular pH and trigger the activation of proton-activated acid-sensing ion channels (ASIC). Claudication pain, linked with low blood flow, can be partially relieved by endogenous opioid peptide release. However, we previously reported that sustained ASIC currents in dorsal root ganglion (DRG) neurons were enhanced by naturally occurring endomorphin-1 and -2 opioid peptides, indicating a role of opioid involvement in hyperalgesia. The present study examined whether clinically employed synthetic (fentanyl, remifentanil) and the semisynthetic opioid (oxycodone) would also potentiate sustained ASIC currents, which arise from ASIC3 channel isoforms. Here, we show that exposure of each opioid to DRG neurons resulted in potentiation of the sustained ASIC currents. On the other hand, the potentiation was not observed in DRG neurons from ASIC3 knockout rats. Further, the enhancement of the ASIC currents was resistant to pertussis toxin treatment, suggesting that Gα i/Gα o G-proteins are not involved. Additionally, the potentiation of sustained ASIC currents was greater in DRG neurons isolated from rats with ligated femoral arteries (a model of PAD). The effect of all three opioids on the transient ASIC peak current was mixed (increase, decrease, no effect). The inhibitory action appears to be mediated by the presence of ASIC1 isoform, while the potentiating effect is primarily due to ASIC3 isoform expression. These findings reveal that, under certain conditions, these three opioids can increase ASIC channel activity, possibly giving rise to opioid-induced hyperalgesia.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Potenciais de Ação/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Animais , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Masculino , Oligopeptídeos/metabolismo , Dor/tratamento farmacológico , Dor/metabolismo , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo
18.
Arch Microbiol ; 201(5): 639-647, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30778632

RESUMO

Many bacteria exploit host proteins for their colonization. Vitronectin (Vn), present in the blood and extracellular matrix, is one such protein that acts as a bridge between the bacteria and the host tissues leading to infection. In this study, Vn binding protein of Staphylococcus aureus (COL strain) (SaVnBP) has been characterized as autolysin(s) based on mass spectrometry data and the ability of these proteins to degrade S. aureus substratum. Deletion of the heparin-binding domain (residues 341-380) from the Vn did not affect its ability to interact with SaVnBP. Similarly, change of R to A or D to A in the second arginine-glycine-aspartic (RGD2) motif of Vn had no negative effect on protein-protein interaction. These results imply that the primary heparin-binding site and the second RGD motif of caprine Vn may not be involved in the initial step of S. aureus colonization.


Assuntos
Proteínas de Transporte/metabolismo , Heparina/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Oligopeptídeos/metabolismo , Staphylococcus aureus/metabolismo , Vitronectina/metabolismo , Animais , Arginina/metabolismo , Ácido Aspártico/metabolismo , Proteínas de Bactérias/metabolismo , Matriz Extracelular , Glicina/metabolismo , Cabras/metabolismo , Humanos , Ligação Proteica/fisiologia , Somatomedinas/metabolismo
19.
J Biotechnol ; 294: 67-72, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30772329

RESUMO

This study focused on a culture system of aerial microalgae with the decomposition of casein protein for obtaining bioactive compounds such as peptides with inhibitory activity against angiotensin-converting enzyme (ACE). The aerial microalga Vischeria helvetica exhibited growth in Bold's basal medium supplemented with casein protein as nitrogen source. The algal cells secreted protease and amino oxidase into the medium, and ammonium ions as a nitrogen source was produced by the conjugated-enzyme reaction. Furthermore, a bioactive peptide with ACE-inhibitory activity was efficiently produced from casein protein by the proteases secreted under light conditions. The results presented will facilitate the development of production systems for useful materials from photosynthetic microorganisms and casein protein in a culture medium.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/metabolismo , Caseínas/metabolismo , L-Aminoácido Oxidase/metabolismo , Microalgas/metabolismo , Oligopeptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Estramenópilas/metabolismo , Proteínas de Algas/metabolismo , Hidrólise , Peptidil Dipeptidase A/metabolismo
20.
Food Chem ; 284: 198-204, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-30744846

RESUMO

A sarcoplasmic serine proteinase (SSP) was purified from threadfin bream (Nemipterus virgatus) belly muscle by ammonium sulfate precipitation and a series of chromatographies including Q-Sepharose, Phenyl Sepharose and Superdex 200. The SSP was purified 1967 folds with a yield of 4.8%. The molecular weight of the SSP was estimated to be 43.5 kDa and 22.5 kDa on SDS-PAGE under non-reducing and reducing conditions, respectively. The N-terminal amino acid sequence of the two protein bands were determined as IVGGYEXQPYSQAHQVSLNSGY and corresponded. It is suggested that the SSP exists as a homodimer. Optimum pH and temperature were 9.5 and 50 °C, using Boc-Val-Pro-Arg-MCA as a substrate. Substrate specificity and effects of inhibitors indicated that the SSP was a trypsin-like serine proteinase. The SSP was responsible for hydrolyzing myosin heavy chain (MHC) and inducing modori phenomenon in the threadfin bream surimi gel. Thus, the SSP was considered as a modori-inducing proteinase.


Assuntos
Peixes , Músculo Esquelético/enzimologia , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismo , Sequência de Aminoácidos , Animais , Cumarínicos/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Peixes da Dieta/química , Proteínas de Peixes da Dieta/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cadeias Pesadas de Miosina/metabolismo , Oligopeptídeos/metabolismo , Inibidores de Serino Proteinase/farmacologia , Especificidade por Substrato , Temperatura Ambiente , Tripsina/metabolismo
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