Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.502
Filtrar
Mais filtros

Intervalo de ano de publicação
1.
Nature ; 582(7813): 592-596, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555458

RESUMO

Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported1, advances in mass-spectrometry-based proteomics2 have enabled increasingly comprehensive identification and quantification of the human proteome3-6. However, there have been few comparisons across species7,8, in stark contrast with genomics initiatives9. Here we use an advanced proteomics workflow-in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system-for the in-depth study of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to the scientific community. The data also provide a large-scale case study for sequence-based machine learning, as we demonstrate by experimentally confirming the predicted properties of peptides from Bacteroides uniformis. Our results offer a comparative view of the functional organization of organisms across the entire evolutionary range. A remarkably high fraction of the total proteome mass in all kingdoms is dedicated to protein homeostasis and folding, highlighting the biological challenge of maintaining protein structure in all branches of life. Likewise, a universally high fraction is involved in supplying energy resources, although these pathways range from photosynthesis through iron sulfur metabolism to carbohydrate metabolism. Generally, however, proteins and proteomes are remarkably diverse between organisms, and they can readily be explored and functionally compared at www.proteomesoflife.org.


Assuntos
Classificação , Aprendizado Profundo , Peptídeos/química , Peptídeos/isolamento & purificação , Proteoma/química , Proteoma/isolamento & purificação , Proteômica/métodos , Animais , Bacteroides/química , Bacteroides/classificação , Metabolismo dos Carboidratos , Cromatografia , Glicólise , Homeostase , Transporte de Íons , Proteínas Ferro-Enxofre/metabolismo , Oxirredução , Fotossíntese , Biossíntese de Proteínas , Dobramento de Proteína , Proteólise , Especificidade da Espécie
2.
J Proteome Res ; 23(8): 3696-3703, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-39013029

RESUMO

We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials, followed by the nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimizing sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5231 protein groups from 500 ng of a HeLa protein digest within 11.5 h (90 min CZE fractionation plus 10 h LC-MS analysis). Finally, the developed platform was used to analyze the protein digest prepared by the MICROFASP method with 1 µg of cell lysate as the starting material. Three thousand seven hundred ninety-six (N = 2, RSD = 4.95%) protein groups and 20,577 (N = 2, RSD = 7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 h. The results indicated that the combination of the MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of a submicrogram biological sample. Data are available via ProteomeXchange with the identifier PXD052735.


Assuntos
Eletroforese Capilar , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Humanos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Eletroforese Capilar/métodos , Células HeLa , Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/análise , Peptídeos/química , Peptídeos/isolamento & purificação , Proteoma/análise , Proteínas/análise , Proteínas/isolamento & purificação , Proteínas/química , Fracionamento Químico/métodos
3.
J Proteome Res ; 23(8): 3484-3495, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-38978496

RESUMO

Data-independent acquisition (DIA) techniques such as sequential window acquisition of all theoretical mass spectra (SWATH) acquisition have emerged as the preferred strategies for proteomic analyses. Our study optimized the SWATH-DIA method using a narrow isolation window placement approach, improving its proteomic performance. We optimized the acquisition parameter combinations of narrow isolation windows with different widths (1.9 and 2.9 Da) on a ZenoTOF 7600 (Sciex); the acquired data were analyzed using DIA-NN (version 1.8.1). Narrow SWATH (nSWATH) identified 5916 and 7719 protein groups on the digested peptides, corresponding to 400 ng of protein from mouse liver and HEK293T cells, respectively, improving identification by 7.52 and 4.99%, respectively, compared to conventional SWATH. The median coefficient of variation of the quantified values was less than 6%. We further analyzed 200 ng of benchmark samples comprising peptides from known ratios ofEscherichia coli, yeast, and human peptides using nSWATH. Consequently, it achieved accuracy and precision comparable to those of conventional SWATH, identifying an average of 95,456 precursors and 9342 protein groups across three benchmark samples, representing 12.6 and 9.63% improved identification compared to conventional SWATH. The nSWATH method improved identification at various loading amounts of benchmark samples, identifying 40.7% more protein groups at 25 ng. These results demonstrate the improved performance of nSWATH, contributing to the acquisition of deeper proteomic data from complex biological samples.


Assuntos
Proteômica , Proteômica/métodos , Humanos , Animais , Camundongos , Células HEK293 , Fígado/metabolismo , Fígado/química , Peptídeos/química , Peptídeos/análise , Peptídeos/isolamento & purificação , Proteoma/análise , Escherichia coli/metabolismo , Escherichia coli/genética , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas/métodos
4.
J Biol Chem ; 299(9): 105066, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37468103

RESUMO

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B. brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11 mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.


Assuntos
Canais de Cálcio Tipo T , Neurotoxinas , Peptídeos , Musaranhos , Animais , Humanos , Sequência de Aminoácidos , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/farmacologia , Peptídeos/síntese química , Peptídeos/genética , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Evolução Molecular , Musaranhos/classificação , Musaranhos/genética , Musaranhos/metabolismo , Tenebrio/efeitos dos fármacos , Células HEK293 , Eletrofisiologia
5.
Anal Chem ; 96(23): 9721-9728, 2024 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-38807522

RESUMO

Can reversed-phase peptide retention be the same for C8 and C18 columns? or increase for otherwise identical columns with a smaller surface area? Can replacing trifluoroacetic acid (TFA) with formic acid (FA) improve the peak shape? According to our common understanding of peptide chromatography, absolutely not. Surprisingly, a thorough comparison of the peptide separation selectivity of 100 and 120 Šfully porous C18 sorbents to maximize the performance of our in-house proteomics LC-MS/MS setup revealed an unexpectedly higher peptide retentivity for a wider pore packing material, despite it having a smaller surface area. Concurrently, the observed increase in peptide retention─which drives variation in separation selectivity between 100 and 120 Špore size materials─was more pronounced for smaller peptides. These findings contradict the central dogmas that underlie the development of all peptide RP-HPLC applications: (i) a larger surface area leads to higher retention and (ii) increasing the pore size should benefit the retention of larger analytes. Based on our intriguing findings, we compared reversed-phase high-performance liquid chromatography peptide retention for a total of 20 columns with pore sizes between 60 and 300 Šusing FA- and TFA-based eluents. Our results unequivocally attest that the larger size of ion pairs in FA- vs TFA-based eluents leads to the observed impact on selectivity and peptide retention. For FA, peptide retention peaks at 200 Špore size, compared to between 120 and 200 Šfor TFA. However, the decrease in retention for narrow-pore particles is more profound in FA. Our findings suggest that common assumptions about analyte size and accessible surface area should be revisited for ion-pair RP separation of small peptides, typical for proteomic applications that are predominantly applying FA eluents. Hybrid silica-based materials with pore sizes of 130-200 Šshould be specifically targeted for bottom-up proteomic applications to obtain both superior peak shape and peptide retentivity. This challenging task of attaining the best RPLC column for proteomics calls for closer collaboration between LC column manufacturers and proteomic LC specialists.


Assuntos
Cromatografia de Fase Reversa , Peptídeos , Proteômica , Proteômica/métodos , Peptídeos/química , Peptídeos/análise , Peptídeos/isolamento & purificação , Porosidade , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Tamanho da Partícula , Ácido Trifluoracético/química , Propriedades de Superfície
6.
Biotechnol Bioeng ; 121(11): 3484-3501, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39099106

RESUMO

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up-to 50-fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.


Assuntos
Cromatografia de Afinidade , Exossomos , Exossomos/química , Exossomos/metabolismo , Cromatografia de Afinidade/métodos , Ligantes , Humanos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/isolamento & purificação
7.
Bioorg Med Chem ; 110: 117831, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39004051

RESUMO

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with Kd estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds.


Assuntos
Antígenos CD20 , Aptâmeros de Nucleotídeos , Eletroforese Capilar , Peptídeos , Técnica de Seleção de Aptâmeros , Humanos , Antígenos CD20/metabolismo , Antígenos CD20/química , Aptâmeros de Nucleotídeos/química , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Peptídeos/isolamento & purificação , Linhagem Celular Tumoral
8.
J Pept Sci ; 30(7): e3572, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38396336

RESUMO

Hairy tofu is a famous Chinese snack that is made from soybeans and rich in various nutrients. In order to further explore the antioxidant peptides of hairy tofu hydrolysates, seven proteases were used to hydrolyze hairy tofu. The results of in vitro radical scavenging activity showed that hairy tofu hydrolysates obtained by pancreatin exhibited the highest antioxidant activity. After Sephadex G-25 gel filtration and reversed-phase high-performance liquid chromatography (RP-HPLC), 97 peptides were identified in the most antioxidant fraction using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Among them, nine peptides were synthesized and their antioxidant activities were assessed using a H2O2-induced oxidative 293T cell model. Finally, four peptides (QCESHK, LAWNEGR, NLQGENEWDQK, and FTEMWR) at concentrations of < 50 µg/ml significantly decreased the malondialdehyde content compared with the model group, displaying in vivo antioxidant activity and low cytotoxicity. Overall, this research provided the choice of using hairy tofu peptides as antioxidant products in the pharmaceutical and food industries.


Assuntos
Antioxidantes , Peptídeos , Humanos , Antioxidantes/química , Antioxidantes/farmacologia , Cromatografia Líquida de Alta Pressão , Células HEK293 , Peróxido de Hidrogênio , Hidrólise , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/isolamento & purificação , Alimentos de Soja/análise
9.
J Nat Prod ; 87(4): 1075-1083, 2024 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-38591246

RESUMO

Cinnamoyl moiety containing nonribosomal peptides represented by pepticinnamin E are a growing family of natural products isolated from different Streptomyces species and possess diverse bioactivities. The soil bacterium Streptomyces mirabilis P8-A2 harbors a cryptic pepticinnamin biosynthetic gene cluster, producing azodyrecins as major products. Inactivation of the azodyrecin biosynthetic gene cluster by CRISPR-BEST base editing led to the activation and production of pepticinnamin E (1) and its analogues, pepticinnamins N, O, and P (2-4), the structures of which were determined by detailed NMR spectroscopy, HRMS data, and Marfey's reactions. These new compounds did not show a growth inhibitory effect against the LNCaP and C4-2B prostate cancer lines, respectively.


Assuntos
Microbiologia do Solo , Streptomyces , Streptomyces/química , Estrutura Molecular , Humanos , Família Multigênica , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/isolamento & purificação , Linhagem Celular Tumoral
10.
Biotechnol Appl Biochem ; 71(3): 627-640, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38311972

RESUMO

Parmotrema perlatum, a lichen belonging to the family Parmeliaceae, is well known for its culinary benefits and aroma used as a condiment in Indian homes is also known as the "black stone flower" or "kalpasi" in India. This research intends to analyze the antioxidant power of the crude extracts using four pH-based buffers solubilized proteins/peptides and RP-HPLC fractions of P. perlatum obtained by purification. The proteins that were extracted from the four different buffers were examined using LC-MS/MS-based peptide mass fingerprinting. When compared to the other buffers, the 0.1 M of Tris-HCl buffer pH 8.0 solubilized proteins/peptides had the strongest antioxidant capacity. The sequential purification of the peptide was carried out by using a 3-kDa cut-off membrane filter and semipreparative RP-HPLC. Additionally, the purified fractions of the peptide's antioxidant activity were assessed, and effects were compared with those of the crude and 3 kDa cut--off membrane filtrates. The peptide fractions were sequenced by LC-MS/MS, which reveals that fraction 2 from RP-HPLC with the sequence LSWFMVVAP has shown the highest antioxidant potential in comparison with other fractions which can serve as the potential natural antioxidant drug. Further, fraction 2 also showed antibacterial activity against the selected microorganisms.


Assuntos
Antibacterianos , Antioxidantes , Espectrometria de Massas em Tandem , Antioxidantes/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Mapeamento de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/isolamento & purificação , Líquens/química , Parmeliaceae/química , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/isolamento & purificação , Espectrometria de Massa com Cromatografia Líquida
11.
J Sep Sci ; 47(15): e2400292, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39091169

RESUMO

This study investigated the capability of electromembrane extraction (EME) as a general technique for peptides, by extracting complex pools of peptides comprising in total of 5953 different substances, varying in size from seven to 16 amino acids. Electromembrane extraction was conducted from a sample adjusted to pH 3.0 and utilized a liquid membrane consisting of 2-nitrophenyl octyl ether and carvacrol (1:1 w/w), containing 2% (w/w) di(2-ethylhexyl) phosphate. The acceptor phase was 50 mM phosphoric acid (pH 1.8), the extraction time was 45 min, and 10 V was used. High extraction efficiency, defined as a higher peptide signal in the acceptor than the sample after extraction, was achieved for 3706 different peptides. Extraction efficiencies were predominantly influenced by the hydrophobicity of the peptides and their net charge in the sample. Hydrophobic peptides were extracted with a net charge of +1, while hydrophilic peptides were extracted when the net charge was +2 or higher. A computational model based on machine learning was developed to predict the extractability of peptides based on peptide descriptors, including the grand average of hydropathy index and net charge at pH 3.0 (sample pH). This research shows that EME has general applicability for peptides and represents the first steps toward in silico prediction of extraction efficiency.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Peptídeos , Peptídeos/química , Peptídeos/isolamento & purificação , Membranas Artificiais , Técnicas Eletroquímicas , Tamanho da Partícula , Concentração de Íons de Hidrogênio , Éteres , Organofosfatos
12.
J Sep Sci ; 47(19): e202400554, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39375913

RESUMO

The increasing awareness of environmental issues and the transition to green analytical chemistry (GAC) have gained popularity among academia and industry in recent years. One of the principles of GAC is the reduction and replacement of toxic solvents with more sustainable and environmentally friendly ones. This review gives an overview of the advances in applying green solvents as an alternative to the traditional organic solvents for peptide and protein purification and analysis by liquid chromatography (LC) and capillary electrophoresis (CE) methods. The feasibility of using greener LC and CE methods is demonstrated through several application examples; however, there is still plenty of room for new developments to fully realize their potential and to address existing challenges. Thanks to the tunable properties of designer solvents, such as ionic liquids and deep eutectic solvents, and almost infinite possible mixtures of components for their production, it is possible that some new designer solvents could potentially surpass the traditional harmful solvents in the future. Therefore, future research should focus mainly on developing new solvent combinations and enhancing analytical instruments to be able to effectively work with green solvents.


Assuntos
Eletroforese Capilar , Química Verde , Peptídeos , Proteínas , Peptídeos/isolamento & purificação , Peptídeos/química , Peptídeos/análise , Proteínas/isolamento & purificação , Proteínas/química , Solventes/química , Cromatografia Líquida/métodos
13.
Curr Microbiol ; 81(10): 319, 2024 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-39167225

RESUMO

With the emergence of multidrug-resistant microorganisms, microbial agents have become a serious global threat, affecting human health and various plants. Therefore, new therapeutic alternatives, such as chitin-binding proteins, are necessary. Chitin is an essential component of the fungal cell wall, and chitin-binding proteins exhibit antifungal activity. In the present study, chitin-binding peptides isolated from Capsicum chinense seeds were characterized and evaluated for their in vitro antimicrobial effect against the growth of Candida and Fusarium fungi. Proteins were extracted from the seeds and subsequently the chitin-binding proteins were separated by chitin affinity chromatography. After chromatography, two fractions, Cc-F1 (not retained on the column) and Cc-F2 (retained on the column), were obtained. Electrophoresis revealed major protein bands between 6.5 and 26.6 kDa for Cc-F1 and only a ~ 6.5 kDa protein band for Cc-F2, which was subsequently subjected to mass spectrometry. The protein showed similarity with hevein-like and endochitinase and was then named Cc-Hev. Data are available via ProteomeXchange with identifier PXD054607. Next, we predicted the three-dimensional structure of the peptides and performed a peptide docking with (NAG)3. Subsequently, growth inhibition assays were performed to evaluate the ability of the peptides to inhibit microorganism growth. Cc-Hev inhibited the growth of C. albicans (up to 75% inhibition) and C. tropicalis (100% inhibition) and induced a 65% decrease in cell viability for C. albicans and 100% for C. tropicalis. Based on these results, new techniques to combat fungal diseases could be developed through biotechnological applications; therefore, further studies are needed.


Assuntos
Antifúngicos , Candida , Capsicum , Quitina , Quitinases , Fusarium , Sementes , Sementes/química , Antifúngicos/farmacologia , Antifúngicos/isolamento & purificação , Antifúngicos/química , Antifúngicos/metabolismo , Quitina/metabolismo , Quitina/farmacologia , Fusarium/efeitos dos fármacos , Quitinases/farmacologia , Quitinases/metabolismo , Quitinases/química , Quitinases/isolamento & purificação , Candida/efeitos dos fármacos , Candida/enzimologia , Lectinas de Plantas/farmacologia , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Simulação de Acoplamento Molecular , Proteínas de Plantas/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Peptídeos Catiônicos Antimicrobianos
14.
Nucleic Acids Res ; 50(2): 651-673, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34967410

RESUMO

Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge-loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge-loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.


Assuntos
Conformação de Ácido Nucleico , Clivagem do RNA , RNA/química , Ribonucleases/química , Sequência de Aminoácidos , Sequência de Bases , Bioensaio/métodos , Catálise , Cinética , Modelos Biológicos , Hibridização de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/isolamento & purificação , Peptídeos/síntese química , Peptídeos/química , Peptídeos/isolamento & purificação , Ribonucleases/metabolismo , Relação Estrutura-Atividade
15.
Mar Drugs ; 22(10)2024 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-39452887

RESUMO

Atopic dermatitis (AD) is a chronic inflammatory skin disorder associated with significant morbidity, including pruritus, recurrent skin lesions, and immune dysregulation. This study aimed to investigate the antioxidative and anti-AD effects of peptides derived from hydrolyzed Sebastes schlegelii (Korea rockfish) tail by-products. Hydrolysates were prepared using various enzymes, including Alcalase, Flavourzyme, Neutrase, and Protamex. Among them, Protamex hydrolysates demonstrated the highest ABTS radical scavenging activity with an RC50 value of 69.69 ± 0.41 µg/mL. Peptides were further isolated from the Protamex hydrolysate using dialysis, fast protein liquid chromatography (FPLC), and high-performance liquid chromatography (HPLC). The most active peptide, STPO-B-II, exhibited a single peak and was identified as a sequence of Glu-Leu-Ala-Lys-Thr-Trp-His-Asp-Met-Lys, designated as MP003. In vivo experiments were conducted using a 2,4-dinitrochlorbenzene (DNCB)-induced AD model in NC/Nga mice. The isolated peptide, MP003, showed significantly reduced AD symptoms, including erythema, lichenification, and collagen deposition. Additionally, MP003 decreased epidermal and dermal thickness, eosinophil, and mast cell infiltration and downregulated the expression of pro-inflammatory cytokines IL-1ß, IL-6, and IgE in serum and skin tissues. These findings suggest that peptides derived from Sebastes schlegelii tail by-products may serve as potential therapeutic agents for AD.


Assuntos
Antioxidantes , Dermatite Atópica , Peptídeos , Animais , Camundongos , Antioxidantes/farmacologia , Antioxidantes/química , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/induzido quimicamente , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/isolamento & purificação , Hidrólise , Modelos Animais de Doenças , Peixes , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Masculino
16.
Mar Drugs ; 22(9)2024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39330294

RESUMO

This study employed a diverse approach to extract antioxidant peptides from red seaweed Palmaria palmata, recognized for its comparatively high protein content. Initially, an aqueous extraction of the entire seaweed was performed, followed by enzymatic hydrolysis of the solid residues prepared from the first step. The effects of three different pH levels (3, 6, and 9) during the aqueous extraction were also examined. Results indicated that the solid fraction from the sequential extraction process contained significantly higher levels of proteins and amino acids than other fractions (p < 0.05). Furthermore, the solid fractions (IC50 ranging from 2.29 to 8.15 mg.mL-1) demonstrated significantly greater free radical scavengers than the liquid fractions (IC50 ranging from 9.03 to 10.41 mg.mL-1 or not obtained at the highest concentration tested) at both stages of extraction (p < 0.05). Among the solid fractions, those produced fractions under alkaline conditions were less effective in radical scavenging than the produced fractions under acidic or neutral conditions. The fractions with most effective metal ion chelating activity were the solid fractions from the enzymatic stage, particularly at pH 3 (IC50 = 0.63 ± 0.04 mg.mL-1) and pH 6 (IC50 = 0.89 ± 0.07 mg.mL-1), which were significantly more effective than those from the initial extraction stage (p < 0.05). Despite no significant difference in the total phenolic content between these solid fractions and their corresponding liquid fractions (3.79 ± 0.05 vs. 3.48 ± 0.02 mg.mL-1 at pH 3 and 2.43 ± 0.22 vs. 2.51 ± 0.00 mg.mL-1 at pH 6) (p > 0.05), the observed antioxidant properties may be attributed to bioactive amino acids such as histidine, glutamic acid, aspartic acid, tyrosine, and methionine, either as free amino acids or within proteins and peptides.


Assuntos
Antioxidantes , Sequestradores de Radicais Livres , Peptídeos , Rodófitas , Alga Marinha , Concentração de Íons de Hidrogênio , Peptídeos/isolamento & purificação , Peptídeos/química , Peptídeos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Antioxidantes/química , Rodófitas/química , Alga Marinha/química , Sequestradores de Radicais Livres/farmacologia , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/química , Aminoácidos/química , Aminoácidos/isolamento & purificação , Hidrólise , Quelantes/farmacologia , Quelantes/química , Quelantes/isolamento & purificação , Algas Comestíveis
17.
Mar Drugs ; 22(9)2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-39330301

RESUMO

For thousands of years, pearl and nacre powders have been important traditional Chinese medicines known for their skin whitening effects. To prepare the enzymatic hydrolysates of Hyriopsis cumingii nacre powder (NP-HCH), complex enzymatic hydrolysis by pineapple protease and of neutral protease was carried out after the powder was pre-treated with a high-temperature and high-pressure method. The peptides were identified using LC-MS/MS and picked out through molecular docking and molecular dynamics simulations. Subsequently, the tyrosinase inhibitory and antioxidant properties of novel tyrosinase inhibitory peptides were investigated in vitro. In addition, the enzymatic activity of tyrosinase in B16F10 cells as well as melanin content and antioxidant enzyme levels were also examined. The results showed that a tyosinase inhibitory peptide (Tyr-Pro-Asn-Pro-Tyr, YPNPY) with an efficient IC50 value of 0.545 ± 0.028 mM was identified. The in vitro interaction results showed that YPNPY is a reversible competitive inhibitor of tyrosinase, suggesting that it binds to the free enzyme. The B16F10 cell whitening test revealed that YPNPY can reduce the melanin content of B16F10 cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that YPNPY could be widely used as a tyrosinase inhibitor in whitening foods and drugs.


Assuntos
Antioxidantes , Melaninas , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase , Peptídeos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Animais , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/isolamento & purificação , Camundongos , Antioxidantes/farmacologia , Antioxidantes/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Linhagem Celular Tumoral , Melanoma Experimental/tratamento farmacológico , Simulação por Computador , Espectrometria de Massas em Tandem , Preparações Clareadoras de Pele/farmacologia , Preparações Clareadoras de Pele/química , Preparações Clareadoras de Pele/isolamento & purificação , Simulação de Dinâmica Molecular
18.
Mar Drugs ; 22(8)2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-39195475

RESUMO

Pearl and nacre powders have been valuable traditional Chinese medicines with whitening properties for thousands of years. We utilized a high-temperature and high-pressure method along with compound enzyme digestion to prepare the enzymatic hydrolysates of nacre powder of Pinctada martensii (NP-PMH). The peptides were identified using LC-MS/MS and screened through molecular docking and molecular dynamics simulations. The interactions between peptides and tyrosinase were elucidated through enzyme kinetics, circular dichroism spectropolarimetry, and isothermal titration calorimetry. Additionally, their inhibitory effects on B16F10 cells were explored. The results showed that a tyrosinase-inhibitory peptide (Ala-His-Tyr-Tyr-Asp, AHYYD) was identified, which inhibited tyrosinase with an IC50 value of 2.012 ± 0.088 mM. The results of the in vitro interactions showed that AHYYD exhibited a mixed-type inhibition of tyrosinase and also led to a more compact enzyme structure. The binding reactions of AHYYD with tyrosinase were spontaneous, leading to the formation of a new set of binding sites on the tyrosinase. The B16F10 cell-whitening assay revealed that AHYYD could reduce the melanin content of the cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that AHYYD could be widely used as a tyrosinase inhibitor in whitening foods and pharmaceuticals.


Assuntos
Inibidores Enzimáticos , Melaninas , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase , Peptídeos , Pinctada , Animais , Monofenol Mono-Oxigenase/antagonistas & inibidores , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/isolamento & purificação , Camundongos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Melaninas/antagonistas & inibidores , Linhagem Celular Tumoral , Melanoma Experimental/tratamento farmacológico , Simulação de Dinâmica Molecular , Simulação por Computador , Antioxidantes/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação
19.
Mar Drugs ; 22(10)2024 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-39452868

RESUMO

Abalone is a rich source of nutrition, the viscera of which are discarded as by-product during processing. This study explored the biological activities of peptides derived from abalone viscera (AV). Trypsin-hydrolysate of AV (TAV) was purified into three fractions using a Sephadex G-10 column. Nine bioactive peptides (VAR, NYER, LGPY, VTPGLQY, QFPVGR, LGEW, QLQFPVGR, LDW, and NLGEW) derived from TAV-F2 were sequenced. LGPY, VTPGLQY, LGEW, LDW, and NLGEW exhibited antioxidant properties, with IC50 values of 0.213, 0.297, 0.289, 0.363, and 0.303 mg/mL, respectively. In vitro analysis determined that the peptides VAR, NYER, VTPGLQY, QFPVGR, LGEW, QLQFPVGR, and NLGEW inhibited ACE, with IC50 values of 0.104, 0.107, 0.023, 0.023, 0.165, 0.004, and 0.146 mg/mL, respectively. The binding interactions of ACE-bioactive peptide complexes were investigated using docking analysis with the ZDCOK server. VTPGLQT interacted with HIS513 and TYR523, and QLQFPVGR interacted with HIS353, ALA354, GLU384, HIS513, and TYR523, contributing to the inhibition of ACE activity. They also interacted with amino acids that contribute to stability by binding to zinc ions. QFPVGR may form complexes with ACE surface sites, suggesting indirect inhibition. These results indicate that AV is a potential source of bioactive peptides with dual antioxidant and anti-hypertensive dual effects.


Assuntos
Inibidores da Enzima Conversora de Angiotensina , Antioxidantes , Gastrópodes , Simulação de Acoplamento Molecular , Peptídeos , Tripsina , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Animais , Antioxidantes/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/isolamento & purificação , Tripsina/química , Gastrópodes/química , Vísceras , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/química , Hidrolisados de Proteína/farmacologia , Hidrolisados de Proteína/química
20.
Mar Drugs ; 22(6)2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38921559

RESUMO

The skin of fish is a physicochemical barrier that is characterized by being formed by cells that secrete molecules responsible for the first defense against pathogenic organisms. In this study, the biological activity of peptides from mucus of Seriola lalandi and Seriolella violacea were identified and characterized. To this purpose, peptide extraction was carried out from epidermal mucus samples of juveniles of both species, using chromatographic strategies for purification. Then, the peptide extracts were characterized to obtain the amino acid sequence by mass spectrometry. Using bioinformatics tools for predicting antimicrobial and antioxidant activity, 12 peptides were selected that were chemically produced by simultaneous synthesis using the Fmoc-Tbu strategy. The results revealed that the synthetic peptides presented a random coil or extended secondary structure. The analysis of antimicrobial activity allowed it to be discriminated that four peptides, named by their synthesis code 5065, 5069, 5070, and 5076, had the ability to inhibit the growth of Vibrio anguillarum and affected the copepodite stage of C. rogercresseyi. On the other hand, peptides 5066, 5067, 5070, and 5077 had the highest antioxidant capacity. Finally, peptides 5067, 5069, 5070, and 5076 were the most effective for inducing respiratory burst in fish leukocytes. The analysis of association between composition and biological function revealed that the antimicrobial activity depended on the presence of basic and aromatic amino acids, while the presence of cysteine residues increased the antioxidant activity of the peptides. Additionally, it was observed that those peptides that presented the highest antimicrobial capacity were those that also stimulated respiratory burst in leukocytes. This is the first work that demonstrates the presence of functional peptides in the epidermal mucus of Chilean marine fish, which provide different biological properties when the fish face opportunistic pathogens.


Assuntos
Aquicultura , Peixes , Muco , Animais , Muco/química , Chile , Antioxidantes/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/isolamento & purificação , Vibrio/efeitos dos fármacos , Epiderme/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA