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1.
World J Microbiol Biotechnol ; 35(9): 133, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432254

RESUMO

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.


Assuntos
Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Virgibacillus/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Genoma Bacteriano , Peso Molecular , Família Multigênica , Peptídeos/química , Peptídeos/genética , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Virgibacillus/classificação , Virgibacillus/isolamento & purificação
2.
Vet Microbiol ; 235: 63-70, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282380

RESUMO

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid (FT) and substantial economic loss in Korea due to egg drop syndrome and mortality. Despite the extensive use of vaccines, FT still occurs in the field. Therefore, the emergence of more pathogenic SG or the recovered pathogenicity of a vaccine strain has been suspected. SpvB, an ADP-ribosyl transferase, is a major pathogenesis determinant, and the length of the polyproline linker (PPL) of SpvB affects pathogenic potency. SG strains accumulate pseudogenes in their genomes during host adaptation, and pseudogene profiling may provide evolutionary information. In this study, we found that the PPL length of Korean SG isolates varied from 11 to 21 prolines and was longer than that of a live vaccine strain, SG 9R (9 prolines). According to growth competition in chickens, the growth of an SG isolate with a PPL length of 17 prolines exceeded that of an SG isolate with a PPL length of 15 prolines. We investigated the pseudogenes of the field isolates, SG 9R and reference strains in GenBank by resequencing and comparative genomics. The pseudogene profiles of the field isolates were notably different from those of the foreign SG strains, and they were subdivided into 7 pseudogene subgroups. Collectively, the field isolates had gradually evolved by changing PPL length and acquiring additional pseudogenes. Thus, the characterization of PPL length and pseudogene profiling may be useful to understand the molecular evolution of SG and the epidemiology of FT.


Assuntos
Galinhas/microbiologia , Evolução Molecular , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/microbiologia , Salmonella enterica/genética , ADP Ribose Transferases/genética , Animais , Surtos de Doenças , Ligantes , Peptídeos/genética , Pseudogenes , República da Coreia , Salmonella enterica/isolamento & purificação , Sorogrupo
3.
Dokl Biochem Biophys ; 485(1): 115-118, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201628

RESUMO

Genetic analysis of thousands of patients with multiple sclerosis (MS) and healthy Russian donors showed that the carriage of groups of HLA-DRB1*15 and HLA-DRB1*03 alleles is associated with the risk of MS, whereas the carriage of groups of HLA-DRB1*01 and HLA-DRB1*11 alleles is protective. Recombinant HLA-DRB1*01:01 with a high affinity can recognize the fragments of myelin basic protein (MBP), one of the autoantigens in MS. However, the comparison of the kinetic parameters of the load of MBP and viral HA peptides on HLA-DRB1*01:01, which is catalyzed by HLA-DM, showed a significantly lower rate of exchange of CLIP for MBP peptides. We assume that the observed protective properties of the group of HLA-DRB1*01 alleles may be directly associated with the ability of HLA-DRB1*01:01 to kinetically distinguish peptides of exogenous and endogenous nature.


Assuntos
Autoantígenos/metabolismo , Cadeias HLA-DRB1/metabolismo , Esclerose Múltipla/metabolismo , Proteína Básica da Mielina/metabolismo , Peptídeos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Feminino , Cadeias HLA-DRB1/química , Cadeias HLA-DRB1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Proteína Básica da Mielina/química , Proteína Básica da Mielina/genética , Peptídeos/química , Peptídeos/genética
4.
Virol J ; 16(1): 75, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159841

RESUMO

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1 h) at - 18 °C, and was compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set. In silico prediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The conditions tested elicited antibodies against the VP1/VP2 antigen that differed in relation to the response time and the profile of structurally available regions that were recognized.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Parvovirus Suíno/imunologia , Animais , Antígenos Virais/química , Mapeamento de Epitopos , Epitopos/química , Masculino , Testes de Neutralização , Peptídeos/genética , Peptídeos/imunologia , Suínos
5.
Nat Commun ; 10(1): 2682, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213602

RESUMO

RNA-protein complexes play essential regulatory roles at nearly all levels of gene expression. Using in vivo crosslinking and RNA capture, we report a comprehensive RNA-protein interactome in a metazoan at four levels of resolution: single amino acids, domains, proteins and multisubunit complexes. We devise CAPRI, a method to map RNA-binding domains (RBDs) by simultaneous identification of RNA interacting crosslinked peptides and peptides adjacent to such crosslinked sites. CAPRI identifies more than 3000 RNA proximal peptides in Drosophila and human proteins with more than 45% of them forming new interaction interfaces. The comparison of orthologous proteins enables the identification of evolutionary conserved RBDs in globular domains and intrinsically disordered regions (IDRs). By comparing the sequences of IDRs through evolution, we classify them based on the type of motif, accumulation of tandem repeats, conservation of amino acid composition and high sequence divergence.


Assuntos
Evolução Molecular , Proteômica/métodos , Motivos de Ligação ao RNA/genética , Proteínas de Ligação a RNA/genética , RNA/metabolismo , Sequência de Aminoácidos/genética , Animais , Linhagem Celular , Sequência Conservada/genética , Reagentes para Ligações Cruzadas/química , Drosophila , Humanos , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Proteoma/genética , RNA/química , Proteínas de Ligação a RNA/química
6.
Cancer Immunol Immunother ; 68(8): 1245-1261, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31222486

RESUMO

The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, often is limited by ineffective presentation of antigenic peptides that elicit T-cell-mediated anti-tumor cytotoxic responses. Manipulation of antigen presentation pathways is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides as part of the system that generates peptides for binding to MHC class I molecules (MHC-I). We hypothesized that pharmacological inhibition of ERAP1 in cells could regulate the cellular immunopeptidome. To test this hypothesis, we treated A375 melanoma cells with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting bound peptides, and identifying them using capillary chromatography and tandem mass spectrometry (LC-MS/MS). Although the inhibitor did not reduce cell-surface MHC-I expression, it induced qualitative and quantitative changes in the presented peptidomes. Specifically, inhibitor treatment altered presentation of about half of the total 3204 identified peptides, including about one third of the peptides predicted to bind tightly to MHC-I. Inhibitor treatment altered the length distribution of eluted peptides without change in the basic binding motifs. Surprisingly, inhibitor treatment enhanced the average predicted MHC-I binding affinity, by reducing presentation of sub-optimal long peptides and increasing presentation of many high-affinity 9-12mers, suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 may be a viable approach for manipulating the immunopeptidome of cancer.


Assuntos
Aminopeptidases/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/metabolismo , Imunoterapia/métodos , Melanoma/tratamento farmacológico , Antígenos de Histocompatibilidade Menor/metabolismo , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Linfócitos T Citotóxicos/imunologia , Aminopeptidases/antagonistas & inibidores , Apresentação do Antígeno , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunogenicidade da Vacina , Ativação Linfocitária , Terapia de Alvo Molecular , Peptídeos/genética , Peptídeos/imunologia , Ligação Proteica
7.
Nat Commun ; 10(1): 2889, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253831

RESUMO

The sinus node is a collection of highly specialised cells constituting the heart's pacemaker. The molecular underpinnings of its pacemaking abilities are debated. Using high-resolution mass spectrometry, we here quantify >7,000 proteins from sinus node and neighbouring atrial muscle. Abundances of 575 proteins differ between the two tissues. By performing single-nucleus RNA sequencing of sinus node biopsies, we attribute measured protein abundances to specific cell types. The data reveal significant differences in ion channels responsible for the membrane clock, but not in Ca2+ clock proteins, suggesting that the membrane clock underpins pacemaking. Consistently, incorporation of ion channel expression differences into a biophysically-detailed atrial action potential model result in pacemaking and a sinus node-like action potential. Combining our quantitative proteomics data with computational modeling, we estimate ion channel copy numbers for sinus node myocytes. Our findings provide detailed insights into the unique molecular make-up of the cardiac pacemaker.


Assuntos
Relógios Biológicos/fisiologia , Peptídeos/química , Peptídeos/metabolismo , Proteômica , Nó Sinoatrial/metabolismo , Transcriptoma , Potenciais de Ação , Animais , Cromatografia Líquida , Regulação da Expressão Gênica/fisiologia , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Espectrometria de Massas em Tandem
8.
Sci Total Environ ; 659: 540-547, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096383

RESUMO

Mercury is a potentially toxic trace metal that poses threats to aquatic life and to humans. In this study, a mercury-binding peptide was displayed on the surface of Escherichia coli cells using an N-terminal region ice nucleation protein anchor. The surface-engineered E. coli facilitated selective adsorption of mercury ions (Hg2+) from a solution containing various metal ions. The Hg2+ adsorption capacity of the surface-engineered cell was four-fold higher than that of the original E. coli cells. Approximately 95% of Hg2+ was removed from solution by these whole-cell sorbents. The transformed strains were fed to Carassius auratus, so that the bacteria could colonize fish intestine. Engineered bacteria-fed C. auratus showed significantly less (51.1%) accumulation of total mercury when compared with the group that had not been fed engineered bacteria. The surface-engineered E. coli effectively protected fish against the toxicity of Hg2+ in aquatic environments by adsorbing more Hg2+. Furthermore, the surface-engineered E. coli mitigated microbial diversity changes in the intestine caused by Hg2+ exposure, thereby protecting the intestinal microbial community. This strategy is a novel approach for controlling Hg2+ contamination in fish.


Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Carpa Dourada/metabolismo , Mercúrio/metabolismo , Peptídeos/metabolismo , Poluentes Químicos da Água/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Engenharia Genética , Mucosa Intestinal/metabolismo , Microrganismos Geneticamente Modificados/genética , Peptídeos/genética
9.
Parasit Vectors ; 12(1): 233, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092283

RESUMO

BACKGROUND: Cryptosporidium parvum is a major cause of diarrhea in children and ruminants at the earliest stages of life. Maternal antibodies represent the main shield of neonate mammals for most of the infections. Two recombinant antigens (SA35 and SA40), portions of two C. parvum proteins, were tested for their ability to induce immune responses in adult mice and for protection on neonate BALB/c mice born from females immunised by mucosal delivery of both peptides. METHODS: Adult BALB/c mice were intraperitoneally immunised with SA35 and SA40, separately or mixed, and their immune response was characterised. Furthermore, BALB/c pregnant mice were immunised by mucosal delivery with an SA35/40 mix, before and during pregnancy. Soon after birth, their offspring were infected with two doses (1 × 105 and 5 × 103) of C. parvum oocysts and the parasitic burden was determined at 5 and 9 days post-infection. RESULTS: Intraperitoneal immunisation with SA35 and SA40 induced specific IgG and IgG1 in serum, specific IgA in the intestinal mucosa, increase of CD3+/CD4+ and CD30+ cells in splenocytes, which produced IFN-γ. Neonates born from immunised mice and infected with 1 × 105 oocysts showed a significant reduction of oocysts and intestinal forms (23 and 42%, respectively). A reduction of all parasitic forms (96%; P < 0.05) was observed when neonates were infected with 5 × 103 oocysts. CONCLUSIONS: SA35 and SA40 peptides induce specific humoral and cell-mediated immune responses to C. parvum in adult mice. Moreover, mucosal administration of the SA35/40 mix in pregnant mice reduces C. parvum burden in their litters.


Assuntos
Criptosporidiose/prevenção & controle , Imunidade Celular , Imunidade Humoral , Peptídeos/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Criptosporidiose/imunologia , Cryptosporidium parvum , Feminino , Imunidade Materno-Adquirida , Imunização , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Mucosa Intestinal/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Oócitos/imunologia , Peptídeos/genética , Gravidez , Proteínas de Protozoários/genética
10.
Molecules ; 24(9)2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31052532

RESUMO

Oceans abound in resources of various kinds for R&D and for commercial applications. Monitoring and bioprospecting allow the identification of an increasing number of key natural resources. Macroalgae are essential elements of marine ecosystems as well as a natural resource influenced by dynamic environmental factors. They are not only nutritionally attractive but have also demonstrated potential health benefits such as antioxidant, antihypertensive, and anti-inflammatory activities. Several bioactive peptides have been observed following enzymatic hydrolysis of macroalgal proteins. In addition, significant differences in protein bioactivities and peptide extracts of wild and cultivated macroalgae have been highlighted, but the metabolic pathways giving rise to these bioactive molecules remain largely elusive. Surprisingly, the biochemistry that underlies the environmental stress tolerance of macroalgae has not been well investigated and remains poorly understood. Proteomic and functional genomic approaches based on identifying precursor proteins and bioactive peptides of macroalgae through integrated multi-omics analysis can give insights into their regulation as influenced by abiotic factors. These strategies allow evaluating the proteomics profile of regulation of macroalgae in response to different growth conditions as well as establishing a comparative transcriptome profiling targeting structural protein-coding genes. Elucidation of biochemical pathways in macroalgae could provide an innovative means of enhancing the protein quality of edible macroalgae. This could be ultimately viewed as a powerful way to drive the development of a tailored production and extraction of high value molecules. This review provides an overview of algal proteins and bioactive peptide characterization using proteomics and transcriptomic analyses.


Assuntos
Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Perfilação da Expressão Gênica , Peptídeos/genética , Peptídeos/metabolismo , Proteoma , Proteômica , Transcriptoma , Perfilação da Expressão Gênica/métodos , Redes e Vias Metabólicas , Proteômica/métodos , Alga Marinha/química , Alga Marinha/genética , Alga Marinha/metabolismo
11.
Chemistry ; 25(43): 10197-10203, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31106456

RESUMO

A simple and efficient strategy for the selective modification of the peptide N terminus with an unnatural amino acid is described. A peptide having a SUMO-HisTag-TEV sequence (SUMO: small ubiquitin-related modifier, TEV: tobacco etch virus) preceding the N terminus of the target peptide was designed. Recombinant expression in E. coli and subsequent SUMO protease cleavage yielded the HisTag-TEV-target peptide. Partial protection of the lysine side chains of this peptide with d-glucopyranosyloxycarbonyl and removal of the HisTag-TEV sequence by TEV protease yielded the partially protected peptide with a free N-terminal amine. Coupling of selenocysteine selectively at the N terminus and subsequent acidic deprotection of the carbohydrate protecting groups yielded a modified peptide that can be used for native chemical ligation (NCL). As a proof of concept, the modification of a longer recombinant peptide with selenocysteinylserine (GalNAc) at the N terminus was demonstrated.


Assuntos
Peptídeos/química , Acetilação , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Endopeptidases/química , Escherichia coli/metabolismo , Histidina/química , Interações Hidrofóbicas e Hidrofílicas , Oligopeptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteína SUMO-1/química , Selenocisteína/química , Espectrometria de Massas por Ionização por Electrospray
12.
Microb Cell Fact ; 18(1): 91, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133014

RESUMO

BACKGROUND: Self-assembling amphipathic peptides (SAPs) may improve protein production or induce the formation of inclusion bodies by fusing them to the N-terminus of proteins. However, they do not function uniformly well with all target enzymes and systematic research on how the composition of SAPs influence the production of fusion protein is still limited. RESULTS: To improve the efficiency of SAPs, we studied factors that might be involved in SAP-mediated protein production using S1 (AEAEAKAK)2 as the original SAP and green fluorescent protein (GFP) as the reporter. The results indicate that hydrophobicity and net charges of SAPs play a key role in protein expression. As hydrophobicity regulation tend to cause the formation of insoluble inclusion bodies of protein, an expression tag library composed of SAPs, which varied in net charge (from + 1 to + 20), was constructed based on the random amplification of S1nv1 (ANANARAR)10. The efficiency of the library was validated by polygalacturonate lyase (PGL), lipoxygenase (LOX), L-asparaginase (ASN) and transglutaminase (MTG). To accelerate preliminary screening, each enzyme was fused at the C-terminus with GFP. Among the four enzyme fusions, the SAPs with + 2 - + 6 net charges were optimal for protein expression. Finally, application of the library improved the expression of PGL, LOX, ASN, and MTG by 8.3, 3.5, 2.64, and 3.68-fold relative to that of the corresponding wild-type enzyme, respectively. CONCLUSIONS: This is the first report to study key factors of SAPs as an expression tag to enhance recombinant enzyme production. The SAP library could be used as a novel plug-and-play protein-engineering method to screen for enzymes or proteins with enhanced production.


Assuntos
Escherichia coli/genética , Biblioteca Gênica , Peptídeos/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas de Fluorescência Verde/química , Ensaios de Triagem em Larga Escala , Interações Hidrofóbicas e Hidrofílicas , Corpos de Inclusão/metabolismo
13.
Science ; 364(6435): 86-89, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948551

RESUMO

Self-recognition is observed abundantly throughout the natural world, regulating diverse biological processes. Although ubiquitous, often little is known of the associated molecular machinery, and so far, organismal self-recognition has never been described in nematodes. We investigated the predatory nematode Pristionchus pacificus and, through interactions with its prey, revealed a self-recognition mechanism acting on the nematode surface, capable of distinguishing self-progeny from closely related strains. We identified the small peptide SELF-1, which is composed of an invariant domain and a hypervariable C terminus, as a key component of self-recognition. Modifications to the hypervariable region, including single-amino acid substitutions, are sufficient to eliminate self-recognition. Thus, the P. pacificus self-recognition system enables this nematode to avoid cannibalism while promoting the killing of competing nematodes.


Assuntos
Canibalismo , Peptídeos/fisiologia , Comportamento Predatório/fisiologia , Rabditídios/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Peptídeos/química , Peptídeos/genética , Domínios Proteicos , Rabditídios/metabolismo , Especificidade da Espécie
14.
Life Sci ; 226: 140-148, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30986446

RESUMO

The surface modification of biomaterials with matrikines for tissue engineering application is one of the recent approaches to improve their biocompatibility. In an earlier study, a peptide containing 21 amino acid isolated from bovine tendon collagen was shown to promote good cell adhesion in HeLa cell, and a smaller region in the peptide was identified using bioinformatics tool to mediate cell-peptide interaction. Hence, the present study was undertaken to validate the cell adhesion property of the smaller region of the peptide and elucidate probable peptide-cell interaction pathway. Cell adhesion and proliferation properties of the peptide were studied on cells cultured on surfaces coated with varying concentrations of peptide. Expression of focal adhesion related proteins like paxillin and pFAK Tyr397 was confirmed by immunoblotting and immunofluorescence microscopy respectively. The anti-pFAK Tyr 397 stained confocal micrographs and mRNA transcription levels of Cdc42 and Rho further confirmed peptide mediated cell spreading. The change in the expression levels of integrin α1 and ß1 indicates an integrin mediated cell-peptide interaction for cell survival and proliferation. Integrin mediated adhesion was further confirmed by anti-integrin blocking assay. The modulation of ECM components by the peptide was assessed by expression of COL1A1, TIMP mRNA levels and gelatin zymography for MMPs. The results of the study confirm the role of the small region of the larger collagen peptide in cell adhesion and proliferation and hint at the possible use of such small peptides as biocompatible surface modifiers for tissue scaffolds.


Assuntos
Adesão Celular/efeitos dos fármacos , Colágeno/fisiologia , Peptídeos/fisiologia , Sequência de Aminoácidos/genética , Aminoácidos , Materiais Biocompatíveis/síntese química , Adesão Celular/fisiologia , Comunicação Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Colágeno/genética , Matriz Extracelular/metabolismo , Fibronectinas , Adesões Focais , Células HeLa , Humanos , Integrinas , Peptídeos/síntese química , Peptídeos/genética , Engenharia Tecidual/métodos
15.
Fish Shellfish Immunol ; 89: 623-631, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30991151

RESUMO

Antimicrobial peptides (AMPs) participate in immune defenses of invertebrate, vertebrate and plant species. As a kind of AMPs, penaeidins play important roles in innate immunity of shrimp. In this study, two penaeidin homologues termed FmPEN3 and FmPEN5 were cloned and identified from Fenneropenaeus merguiensis for the first time. The complete open reading frames (ORFs) of FmPEN3 and FmPEN5 were 216 bp and 240 bp, encoding 71 and 79 amino acids, respectively. Both FmPEN3 and FmPEN5 contain an N-terminal proline-rich domain (PRD) and a C-terminal cysteine-rich domain (CRD). The genome structure of FmPEN3 and FmPEN5 genes both consist of 2 exons and 1 intron. qPCR analysis showed that FmPEN3 was constitutively expressed but FmPEN5 transcripts were found only in hemocytes, gills, epidermis, nerve and pyloric cecum. The FmPEN3 and FmPEN5 expression were responsive to Vibrio parahaemolyticus and Micrococcus lysodeikticus infection and their transcription levels were downregulated by RNAi silencing of the transcription factors FmDorsal and FmRelish. In addition, recombinant proteins of FmPEN3 (rFmPEN3) and FmPEN5 (rFmPEN5) were successfully expressed in E. coli. The antibacterial assays revealed that rFmPEN3 and rFmPEN5 could inhibit the growth of M. lysodeikticus but only rFmPEN5 could inhibit the growth of V. parahaemolyticus in vitro. In summary, the results presented in this study indicated the functions of FmPEN3 and FmPEN5 played in anti-bacterial immunity of F. merguiensis, providing some insights into the function of AMPs in shrimp.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Peptídeos/genética , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Micrococcus/fisiologia , Peptídeos/química , Filogenia , Alinhamento de Sequência , Vibrio parahaemolyticus/fisiologia
16.
Molecules ; 24(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022909

RESUMO

Fused in sarcoma (FUS) is a DNA/RNA binding protein that is involved in RNA metabolism and DNA repair. Numerous reports have demonstrated by pathological and genetic analysis that FUS is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and polyglutamine diseases. Traditionally, the fibrillar aggregation of FUS was considered to be the cause of those diseases, especially via its prion-like domains (PrLDs), which are rich in glutamine and asparagine residues. Lately, a nonfibrillar self-assembling phenomenon, liquid-liquid phase separation (LLPS), was observed in FUS, and studies of its functions, mechanism, and mutual transformation with pathogenic amyloid have been emerging. This review summarizes recent studies on FUS self-assembling, including both aggregation and LLPS as well as their relationship with the pathology of ALS, FTLD, and other neurodegenerative diseases.


Assuntos
Doenças Neurodegenerativas/genética , Agregação Patológica de Proteínas/genética , Proteína FUS de Ligação a RNA/química , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Asparagina/química , Asparagina/genética , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/patologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Humanos , Doenças Neurodegenerativas/patologia , Peptídeos/química , Peptídeos/genética , Príons/química , Príons/genética , Agregação Patológica de Proteínas/patologia , Domínios Proteicos/genética , Proteína FUS de Ligação a RNA/genética
17.
J Mol Model ; 25(5): 112, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953170

RESUMO

Membranolytic anticancer peptides (ACPs) are drawing increasing attention as potential future therapeutics against cancer, due to their ability to hinder the development of cellular resistance and their potential to overcome common hurdles of chemotherapy, e.g., side effects and cytotoxicity. In this work, we present an ensemble machine learning model to design potent ACPs. Four counter-propagation artificial neural-networks were trained to identify peptides that kill breast and/or lung cancer cells. For prospective application of the ensemble model, we selected 14 peptides from a total of 1000 de novo designs, for synthesis and testing in vitro on breast cancer (MCF7) and lung cancer (A549) cell lines. Six de novo designs showed anticancer activity in vitro, five of which against both MCF7 and A549 cell lines. The novel active peptides populate uncharted regions of ACP sequence space.


Assuntos
Antineoplásicos/química , Modelos Moleculares , Neoplasias/tratamento farmacológico , Peptídeos/química , Células A549 , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Humanos , Células MCF-7 , Aprendizado de Máquina , Neoplasias/genética , Redes Neurais (Computação) , Peptídeos/genética , Peptídeos/uso terapêutico
18.
Bioengineered ; 10(1): 87-97, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30957636

RESUMO

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)20, was engineered either at the N- or C-terminal end of AAT. The (AP)20 tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)20-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)20 along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)20 module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.


Assuntos
Elastase Pancreática/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , alfa 1-Antitripsina/biossíntese , Sequência de Bases , Técnicas de Cultura de Células , Dipeptídeos/genética , Dipeptídeos/metabolismo , Expressão Gênica , Glicosilação , Humanos , Elastase Pancreática/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Células Vegetais/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tabaco/citologia , Tabaco/metabolismo , Transformação Genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/farmacologia
19.
Biomed Res Int ; 2019: 7084734, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30941370

RESUMO

Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whether i.v. butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins, and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins (ZO-1, claudin-7, E-cadherin), antimicrobial peptides (Defa 8, Rd5, RegIIIγ), and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10, and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/pathogenic genera, which might contribute to its overall beneficial effect.


Assuntos
Butiratos/farmacologia , Suplementos Nutricionais , Microbioma Gastrointestinal , Intestinos/patologia , Nutrição Parenteral , Animais , Biodiversidade , Colo/efeitos dos fármacos , Colo/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Íleo/efeitos dos fármacos , Íleo/patologia , Intestino Delgado/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Modelos Animais , Mucinas/biossíntese , Celulas de Paneth/efeitos dos fármacos , Celulas de Paneth/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Permeabilidade , Fenótipo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Proteínas de Junções Íntimas/metabolismo
20.
Int J Mol Sci ; 20(8)2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003532

RESUMO

Antigen-mimicking peptide (mimotope)-based vaccines are one of the most promising forms of active-immunotherapy. The main drawback of this approach is that it induces antibodies that react poorly with the nominal antigen. The aim of this study was to investigate the molecular basis underlying the weak antibody response induced against the naïve protein after peptide vaccination. For this purpose, we analyzed the fine specificity of monoclonal antibodies (mAb) elicited with a 13-mer linear peptide, complementary to theantigen-combining site of the anti-CD20 mAb, Rituximab, in BALB/c mice. Anti-peptide mAb competed with Rituximab for peptide binding. Even so, they recognized a different antigenic motif from the one recognized by Rituximab. This explains their lack of reactivity with membrane (naïve) CD20. These data indicate that even on a short peptide the immunogenic and antigenic motifs may be different. These findings highlight an additional mechanism for epitope spreading and should be taken into account when designing peptides for vaccine purposes.


Assuntos
Anticorpos Monoclonais/genética , Antígenos CD20/imunologia , Peptídeos/genética , Rituximab/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos/genética , Anticorpos Monoclonais Murinos/imunologia , Antígenos CD20/genética , Sítios de Ligação de Anticorpos/genética , Epitopos/genética , Epitopos/imunologia , Humanos , Camundongos , Biblioteca de Peptídeos , Peptídeos/imunologia , Rituximab/genética , Vacinação/métodos , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia
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