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1.
BMC Genomics ; 22(1): 640, 2021 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-34481473

RESUMO

BACKGROUND: Fatty liver disease prevalently occurs in commercial postpartum dairies, resulting in a worldwide high culling rate because of their subsequent limitations of production and reproduction performance. RESULTS: Fatty liver-specific proteome and acetylome analysis revealed that energy metabolism suppression closely associated with mitochondrial dysfunction and inflammation activation were shown to be remarkable biological processes underlying the development of fatty liver disease, furthermore, acetylation modification of proteins could be one of the main means to modulate these processes. Twenty pivotal genetic factors/genes that differentially expressing and being acetylation modified in liver were identified and proposed to regulate the pathogenesis of fatty liver dairies. These proteins were confirmed to be differentially expressing in individual liver tissue, eight of which being validated via immunohistochemistry assay. CONCLUSIONS: This study provided a comprehensive proteome and acetylome profile of fatty liver of dairy cows, and revealed potential important biological processes and essential regulators in the pathogenesis of fatty liver disease. Expectantly, understanding the molecular mechanisms of the pathogenesis of fatty liver disease in dairies, as an animal model of non-alcoholic fatty liver disease (NAFLD) in human beings, which is a clinico-pathologically defined process associated with metabolic syndrome, could inspire and facilitate the development of efficacious therapeutic drugs on NAFLD.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Proteoma , Animais , Bovinos , Feminino , Humanos , Inflamação , Mitocôndrias , Hepatopatia Gordurosa não Alcoólica/genética
2.
Mater Sci Eng C Mater Biol Appl ; 128: 112289, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34474840

RESUMO

Successful osseointegration, i.e. the fully functional connection of patient's bone and artificial implant depends on the response of the cells to the direct contact with the surface of the implant. The surface properties of the implant which trigger cell responses leading to its integration into the surrounding bone can be tailored by surface modifications or coating with thin layers. One potential material for such applications is ultrananocrystalline diamond (UNCD). It combines the exceptional mechanical properties of diamond with good biocompatibility and possibility of coating as thin uniform films on different substrates of biological interest. In the current work we firstly deposited UNCD films on titanium-coated substrates and applied oxygen or ammonia plasma to modify their surface properties. The as-grown and modified UNCD exhibited relatively smooth surfaces with topography dominated by rounded features. The modifications induced oxygen- or amino-terminated surfaces with increased hydrophilicity. In addition, the UNCD coatings exhibited very low coefficient of friction when diamond was used as a counterpart. As-grown and modified UNCD samples were applied to study the responses of human osteoblast MG63 cells triggered by surfaces with various terminations assessed by proteomic analysis. The results revealed that the coating of Ti with UNCD as well as the plasma modifications resulting in O- or NH2-terminated UNCD induced upregulation of proteins specific for cytoskeleton, cell membrane, and extracellular matrix (ECM) involved in the cell-ECM-surface interactions. Proteins from each of these groups, namely, vimentin, cadherin and fibronectin were further studied immunocytochemically and the results confirmed their increased abundance leading to improved cell-to-surface adhesion and cell-to-cell interactions. These findings demonstrate the potential of implant coating with UNCD and its surface modifications for better osseointegration and bone formation.


Assuntos
Proteoma , Titânio , Diamante , Humanos , Osteoblastos , Proteômica , Propriedades de Superfície
3.
BMC Genomics ; 22(1): 648, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493209

RESUMO

BACKGROUND: Bacillus cereus is a notorious foodborne pathogen, which can grow under anoxic conditions. Anoxic growth is supported by endogenous redox metabolism, for which the thiol redox proteome serves as an interface. Here, we studied the cysteine (Cys) proteome dynamics of B. cereus ATCC 14579 cells grown under fermentative anoxic conditions. We used a quantitative thiol trapping method combined with proteomics profiling. RESULTS: In total, we identified 153 reactive Cys residues in 117 proteins participating in various cellular processes and metabolic pathways, including translation, carbohydrate metabolism, and stress response. Of these reactive Cys, 72 were detected as reduced Cys. The B. cereus Cys proteome evolved during growth both in terms of the number of reduced Cys and the Cys-containing proteins identified, reflecting its growth-phase-dependence. Interestingly, the reduced status of the B. cereus thiol proteome increased during growth, concomitantly to the decrease of extracellular oxidoreduction potential. CONCLUSIONS: Taken together, our data show that the B. cereus Cys proteome during unstressed fermentative anaerobic growth is a dynamic entity and provide an important foundation for future redox proteomic studies in B. cereus and other organisms.


Assuntos
Bacillus cereus , Proteoma , Anaerobiose , Oxirredução , Proteoma/metabolismo , Proteômica , Compostos de Sulfidrila
4.
J Agric Food Chem ; 69(36): 10731-10740, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34469689

RESUMO

Although the antimicrobial, nutritional, and health-promoting properties of royal jelly (RJ) have been widely confirmed, the effects of storage temperature and time on RJ quality remain to be further explored. Herein, the antimicrobial and proteomic dynamics of RJ stored under different conditions were comprehensively investigated to identify consistent and sensitive markers of RJ degradation. We confirmed the negative correlation between antimicrobial properties and increased the storage temperature and duration in RJ. Using surface plasmon resonance, we showed the protein degradation-induced conformation changes in RJ, which reflected the overall variation in RJ proteins caused by the storage conditions. Further proteomic and western blotting analyses demonstrated the sensitivity and reliability of major RJ protein 4 (MRJP4) as a measure of temperature- and time-dependent RJ changes. Based on these results, we developed a colloidal gold immunoassay strip for MRJP4 detection, providing a reliable, simple, and rapid method for the evaluation of RJ freshness.


Assuntos
Anti-Infecciosos , Proteoma , Anti-Infecciosos/farmacologia , Ácidos Graxos , Proteômica , Reprodutibilidade dos Testes , Temperatura
5.
J Agric Food Chem ; 69(36): 10749-10759, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34474557

RESUMO

Gestational diabetes mellitus (GDM) not only has a bad effect on the development of infants but also causes variations in breastmilk composition. This study aims to investigate the changes in the protein profile of colostrum between mothers with GDM and healthy mothers (H) by sequential windowed acquisition of all theoretical fragment ion proteomics techniques. A total of 1295 proteins were detected, with 192 proteins being significantly different between GDM and H. These significantly different proteins were enriched with the carbohydrate and lipid metabolism pathway as well as immunity. Some proteins had an AOC value of 1, such as apolipoprotein E and lipoprotein lipase. In addition, we identified 42 glycated and 93 glycosylated peptides in colostrum without any enrichment, with glycated peptides being upregulated and glycosylated peptides being downregulated in colostrum with GDM. These results help us to better understand the GDM-induced changes in proteomes and glycated and glycosylated level and provide guidance on infant formula adjustment for infants from mothers with GDM.


Assuntos
Diabetes Gestacional , Colostro , Feminino , Humanos , Leite Humano , Gravidez , Proteoma , Proteômica
6.
Nanoscale ; 13(31): 13353-13367, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34477741

RESUMO

Despite the significance of surface absorbed proteins in determining the biological identity of nanoparticles (NPs) entering the human body, little is known about the surface corona and factors that shape their formation on dietary particles used as food additives. In this study, food grade NPs of silica and titania and their food additive counterparts (E551 and E171) were interacted with milk proteins or with skimmed milk and the levels of protein adsorption were quantified. Characteristics of proteins correlating with their level of adsorption to NPs were determined using partial least squares regression analysis. Results from individual protein-particle interactions revealed the significance of factors such as zeta potential, hydrophobicity and hydrodynamic size of particles, and protein characteristics such as the number of beta strands, isoelectric points, the number of amino acid units (Ile, Tyr, Ala, Gly, Pro, Asp, and Arg), and phosphorylation sites on their adsorption to particles. Similar regression analysis was performed to identify the characteristics of twenty abundant and enriched proteins (identified using LC-MS/MS analysis) for their association with the surface corona of milk-interacted particles. Contrary to individual protein-particle interactions, protein characteristics such as helices, turns, protein structures, disulfide bonds, the number of amino acid units (Cys, Met, Leu, and Trp), and Fe binding sites were significant for their association with the surface corona of milk interacted particles. This difference in factors identified from individual proteins and milk interacted particles suggested possible interactions of proteins with surface adsorbed biomolecules as revealed by scanning transmission X-ray microscopy and other biochemical assays.


Assuntos
Nanopartículas , Coroa de Proteína , Adsorção , Sequência de Aminoácidos , Cromatografia Líquida , Humanos , Proteoma , Espectrometria de Massas em Tandem
7.
Se Pu ; 39(9): 981-988, 2021 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-34486837

RESUMO

Protein glycosylation is among the most common and important post-translational modifications, and plays an important regulatory role in many biological processes, including signal transduction, protein translation, and immune response. Abnormal protein glycosylation is also associated with numerous diseases, suggesting that glycoproteins may offer an array of useful disease biomarkers. Mass spectrometry (MS) has become an important analytical tool in glycoproteomics. However, the low abundance and weak ionization efficiency of glycopeptides have hindered direct mass spectrometric analyses, which remain considerably challenging. Glycoprotein and glycopeptide enrichment from complex biological samples is an important step in glycoproteomics. Diverse methods have recently been developed for specific glycoprotein and glycopeptide enrichment, including hydrophilic interaction liquid chromatography (HILIC), lectin affinity chromatography, boronate affinity chromatography, and hydrazide functional affinity chromatography. A variety of enrichment materials designed for the above strategies have been developed to meet the requirement of enriching low abundance glycoproteins and glycopeptides in complex samples. Magnetic solid phase extraction (MSPE) is an efficient sample pretreatment technology that offers advantages of simple operation, low cost, and high extraction efficiency. Functionalized magnetic nanomaterials have been widely used as adsorbents in glycoproteome studies. Since magnetic adsorbent is a key factor in MSPE, in this review, the preparation of magnetic nanomaterials functionalized with sugars, ionic liquids, lectins, boronate affinity ligands, metal organic frameworks, and covalent organic frameworks, and their applications in glycoprotein and glycopeptide enrichment are summarized. These functional magnetic nanomaterials possess high specific surface area and a large number of active adsorption sites, allowing different enrichment mechanisms, including HILIC, lectin affinity chromatography, and boronate and hydrazide functional affinity chromatography. These functional magnetic nanomaterials are mainly used to enrich glycoproteins and glycopeptides in serum, plasma, cells, tissues, saliva and other biological samples. Nearly 90 papers published in the last decade from the Science Citation Index (SCI) and Chinese core journals have been cited in this paper. Finally, the development and prospects of magnetic nanomaterials in glycoprotein and glycopeptide enrichment are also discussed.


Assuntos
Glicopeptídeos , Nanoestruturas , Glicoproteínas , Interações Hidrofóbicas e Hidrofílicas , Fenômenos Magnéticos , Proteoma
8.
Curr Protoc ; 1(9): e245, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34516047

RESUMO

Studies in various tissues have revealed a central role of metabolic pathways in regulating adult stem cell function in tissue regeneration and tumor initiation. The unique metabolic dependences or preferences of adult stem cells, therefore, are emerging as a new category of therapeutic target. Recently, advanced methods including high-resolution metabolomics, proteomics, and transcriptomics have been developed to address the growing interest in stem cell metabolism. A practical framework integrating the omics analyses is needed to systematically perform metabolic characterization in a cell-type-specific manner. Here, we leverage recent advances in transcriptomics and proteomics research to identify cell-type-specific metabolic features by reconstructing cell identity using genes and the encoded enzymes involved in major metabolic pathways. We provide protocols for cell isolation, transcriptome and proteome analyses, and metabolite profiling and measurement. The workflow for mapping cell-type-specific metabolic signatures presented here, although initially developed for intestinal crypt cells, can be easily implemented for cell populations in other tissues, and is highly compatible with most public datasets. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Intestinal crypt isolation and cell population purification Basic Protocol 2: Transcriptome analyses for cell-type-specific metabolic gene expression Basic Protocol 3: Proteome analyses for cell-type-specific metabolic enzyme levels Basic Protocol 4: Metabolite profiling and measurement.


Assuntos
Proteoma , Transcriptoma , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/genética , Proteoma/genética , Proteômica
9.
Anal Chem ; 93(36): 12312-12319, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34469131

RESUMO

Direct infusion shotgun proteome analysis (DISPA) is a new paradigm for expedited mass spectrometry-based proteomics, but the original data analysis workflow was onerous. Here, we introduce CsoDIAq, a user-friendly software package for the identification and quantification of peptides and proteins from DISPA data. In addition to establishing a complete and automated analysis workflow with a graphical user interface, CsoDIAq introduces algorithmic concepts to spectrum-spectrum matching to improve peptide identification speed and sensitivity. These include spectra pooling to reduce search time complexity and a new spectrum-spectrum match score called match count and cosine, which improves target discrimination in a target-decoy analysis. Fragment mass tolerance correction also increased the number of peptide identifications. Finally, we adapt CsoDIAq to standard LC-MS DIA and show that it outperforms other spectrum-spectrum matching software.


Assuntos
Proteoma , Software , Algoritmos , Cromatografia Líquida , Bases de Dados de Proteínas , Proteômica
10.
Nat Commun ; 12(1): 5204, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471136

RESUMO

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.


Assuntos
Retículo Endoplasmático/metabolismo , Canais de Translocação SEC/metabolismo , Via Secretória/fisiologia , Animais , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Proteoma/metabolismo , Proteômica
11.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360905

RESUMO

Some Listeria species are important human and animal pathogens that can be found in contaminated food and produce a variety of virulence factors involved in their pathogenicity. Listeria strains exhibiting multidrug resistance are known to be progressively increasing and that is why continuous monitoring is needed. Effective therapy against pathogenic Listeria requires identification of the bacterial strain involved, as well as determining its virulence factors, such as antibiotic resistance and sensitivity. The present study describes the use of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to do a global shotgun proteomics characterization for pathogenic Listeria species. This method allowed the identification of a total of 2990 non-redundant peptides, representing 2727 proteins. Furthermore, 395 of the peptides correspond to proteins that play a direct role in Listeria pathogenicity; they were identified as virulence factors, toxins and anti-toxins, or associated with either antibiotics (involved in antibiotic-related compounds production or resistance) or resistance to toxic substances. The proteomic repository obtained here can be the base for further research into pathogenic Listeria species and facilitate the development of novel therapeutics for these pathogens.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana Múltipla , Listeria/efeitos dos fármacos , Listeria/patogenicidade , Proteoma/química , Fatores de Virulência/química , Transportadores de Cassetes de Ligação de ATP/química , Cromatografia Líquida/métodos , Genes Bacterianos , Listeria/classificação , Listeria/genética , Peptídeos/química , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
12.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360914

RESUMO

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant's best start at a healthy life. One key component of human milk is ß-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk's endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of ß-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of ß-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in ß-casein's known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of ß-casein may be important as it resides on known ß-casein-derived antimicrobial peptide sequences.


Assuntos
Caseínas/metabolismo , Glicopeptídeos/química , Lactação/metabolismo , Leite Humano/química , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/química , Aleitamento Materno , Cromatografia Líquida/métodos , Feminino , Glicosilação , Voluntários Saudáveis , Humanos , Lactente , Estudos Longitudinais , Fosforilação , Espectrometria de Massas em Tandem/métodos
13.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361776

RESUMO

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.


Assuntos
Antineoplásicos/química , Antioxidantes/química , Antivirais/química , Proteínas Fúngicas/química , Pleurotus/química , Proteoma/química , Cogumelos Shiitake/química , Aminoácidos/química , Aminoácidos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Antivirais/isolamento & purificação , Antivirais/farmacologia , Benzotiazóis/antagonistas & inibidores , Compostos de Bifenilo/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Misturas Complexas/química , Flavonoides/química , Flavonoides/isolamento & purificação , Proteínas Fúngicas/classificação , Proteínas Fúngicas/isolamento & purificação , Humanos , Lectinas/química , Lectinas/isolamento & purificação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Especificidade de Órgãos , Fenóis/química , Fenóis/isolamento & purificação , Picratos/antagonistas & inibidores , Pleurotus/metabolismo , Cultura Primária de Células , Proteoma/classificação , Proteoma/isolamento & purificação , Serina Proteases/química , Serina Proteases/isolamento & purificação , Cogumelos Shiitake/metabolismo , Ácidos Sulfônicos/antagonistas & inibidores , Superóxido Dismutase/química , Superóxido Dismutase/isolamento & purificação , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/isolamento & purificação , Vitaminas/química , Vitaminas/isolamento & purificação , Água/química
14.
Anal Chem ; 93(35): 11946-11955, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34431655

RESUMO

Chemical proteomics is widely used for the global investigation of protein activity and binding of small molecule ligands. Covalent probe binding and inhibition are assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to gain molecular information on targeted proteins and probe-modified sites. The identification of amino acid sites modified by large complex probes, however, is particularly challenging because of the increased size, hydrophobicity, and charge state of peptides derived from modified proteins. These studies are important for direct evaluation of proteome-wide selectivity of inhibitor scaffolds used to develop targeted covalent inhibitors. Here, we disclose reverse-phase chromatography and MS dissociation conditions tailored for binding site identification using a clickable covalent kinase inhibitor containing a sulfonyl-triazole reactive group (KY-26). We applied this LC-MS/MS strategy to identify tyrosine and lysine sites modified by KY-26 in functional sites of kinases and other ATP-/NAD-binding proteins (>65 in total) in live cells. Our studies revealed key bioanalytical conditions to guide future chemical proteomic workflows for direct target site identification of complex irreversible probes and inhibitors.


Assuntos
Proteômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Proteoma , Triazóis
15.
Front Immunol ; 12: 669357, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34349756

RESUMO

Development of adaptive immunity after COVID-19 and after vaccination against SARS-CoV-2 is predicated on recognition of viral peptides, presented on HLA class II molecules, by CD4+ T-cells. We capitalised on extensive high-resolution HLA data on twenty five human race/ethnic populations to investigate the role of HLA polymorphism on SARS-CoV-2 immunogenicity at the population and individual level. Within populations, we identify wide inter-individual variability in predicted peptide presentation from structural, non-structural and accessory SARS-CoV-2 proteins, according to individual HLA genotype. However, we find similar potential for anti-SARS-CoV-2 cellular immunity at the population level suggesting that HLA polymorphism is unlikely to account for observed disparities in clinical outcomes after COVID-19 among different race/ethnic groups. Our findings provide important insight on the potential role of HLA polymorphism on development of protective immunity after SARS-CoV-2 infection and after vaccination and a firm basis for further experimental studies in this field.


Assuntos
COVID-19/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Imunidade Celular , SARS-CoV-2/imunologia , Apresentação do Antígeno , Linfócitos T CD4-Positivos/imunologia , COVID-19/genética , Genótipo , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Peptídeos/imunologia , Polimorfismo Genético , Proteoma/imunologia , Proteínas Virais/imunologia
16.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361073

RESUMO

This study evaluated the immunonutritional effects caused by protease inhibitors from Avena sativa and Triticum durum to human macrophage-like cells. Macrophages were exposed (3 h) to extracts obtained from flours, and mitochondrial-associated oxygen consumption rates and inflammatory, metabolic, and proteome adaptations were quantified. Mass spectrometry 'm/z' signals of the extracts obtained from T. durum and A. sativa revealed molecular weights of 18-35 kDa and 16-22 kDa, respectively, for the compounds present at highest concentrations. Extracts from T. durum exhibited lower susceptibility to degradation by gastrointestinal enzymes than those from A. sativa: 9.5% vs 20.2%. Despite their different botanical origin, both extracts increased TLR4 expression. Metabolic protein levels were indicative of a decreased glycolytic to lactate flux in cell cultures upon stimulation with A. sativa extracts, which improved mitochondrial respiration in relation to those from T. durum. Principal components analysis confirmed relative similarities between immune-metabolic events triggered by immunonutritional ingredients in T. durum and A. sativa. Collectively, immunonutritional effects help to interpret the differences between both crops, worsening or improving, macrophage immune reactivity (tolerogenicity), and better control of inflammatory processes.


Assuntos
Avena/química , Macrófagos/imunologia , Macrófagos/metabolismo , Extratos Vegetais/farmacologia , Inibidores de Proteases/farmacologia , Proteoma/efeitos dos fármacos , Triticum/química , Humanos , Macrófagos/efeitos dos fármacos
17.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360666

RESUMO

The ability to fertilise an egg is acquired by the mammalian sperm during the complex biochemical process called capacitation. Capacitation is accompanied by the production of reactive oxygen species (ROS), but the mechanism of redox regulation during capacitation has not been elucidated. This study aimed to verify whether capacitation coincides with reversible oxidative post-translational modifications of proteins (oxPTMs). Flow cytometry, fluorescence microscopy and Western blot analyses were used to verify the sperm capacitation process. A fluorescent gel-based redox proteomic approach allowed us to observe changes in the level of reversible oxPTMs manifested by the reduction or oxidation of susceptible cysteines in sperm proteins. Sperm capacitation was accompanied with redox modifications of 48 protein spots corresponding to 22 proteins involved in the production of ROS (SOD, DLD), playing a role in downstream redox signal transfer (GAPDHS and GST) related to the cAMP/PKA pathway (ROPN1L, SPA17), acrosome exocytosis (ACRB, sperm acrosome associated protein 9, IZUMO4), actin polymerisation (CAPZB) and hyperactivation (TUBB4B, TUB1A). The results demonstrated that sperm capacitation is accompanied by altered levels of oxPTMs of a group of redox responsive proteins, filling gaps in our knowledge concerning sperm capacitation.


Assuntos
Reação Acrossômica , Exocitose , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Capacitação Espermática , Animais , Bovinos , Fertilização , Masculino , Oxirredução , Fosforilação , Proteoma/análise , Proteoma/química
18.
Curr Protoc ; 1(8): e213, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34370893

RESUMO

Protein methyltransferases (PMTs) regulate many aspects of normal and disease processes through substrate methylation, with S-adenosyl-L-methionine (SAM) as a cofactor. It has been challenging to elucidate cellular protein lysine and arginine methylation because these modifications barely alter physical properties of target proteins and often are context dependent, transient, and substoichiometric. To reveal bona fide methylation events associated with specific PMT activities in native contexts, we developed the live-cell Bioorthogonal Profiling of Protein Methylation (lcBPPM) technology, in which the substrates of specific PMTs are labeled by engineered PMTs inside living cells, with in situ-synthesized SAM analogues as cofactors. The biorthogonality of this technology is achieved because these SAM analogue cofactors can only be processed by the engineered PMTs-and not native PMTs-to modify the substrates with distinct chemical groups. Here, we describe the latest lcBPPM protocol and its application to reveal proteome-wide methylation and validate specific methylation events. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live-cell labeling of substrates of protein methyltransferases GLP1 and PRMT1 with lcBPPM-feasible enzymes and SAM analogue precursors Support Protocol: Gram-scale synthesis of Hey-Met Basic Protocol 2: Click labeling of lcBPPM cell lysates with a biotin-azide probe Alternate Protocol: Click labeling of small-scale lcBPPM cell lysates with a TAMRA-azide dye for in-gel fluorescence visualization Basic Protocol 3: Enrichment of biotinylated lcBPPM proteome with streptavidin beads Basic Protocol 4: Proteome-wide identification of lcBPPM targets with mass spectrometry Basic Protocol 5: Validation of individual lcBPPM targets by western blot.


Assuntos
Metionina , S-Adenosilmetionina , Humanos , Metilação , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteoma/metabolismo , Proteínas Repressoras , S-Adenosilmetionina/metabolismo
19.
Nat Commun ; 12(1): 4696, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349113

RESUMO

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Temperatura Baixa , GTP Fosfo-Hidrolases/metabolismo , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoma/metabolismo , Dobramento de RNA , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
20.
Nat Commun ; 12(1): 4669, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344873

RESUMO

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.


Assuntos
Sistema Nervoso Central/metabolismo , Chaperonas Moleculares/metabolismo , Mapeamento de Interação de Proteínas/instrumentação , Proteoma/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Glioblastoma/diagnóstico , Glioblastoma/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Sondas Moleculares/química , Sondas Moleculares/farmacocinética , Sondas Moleculares/farmacologia , Sondas Moleculares/uso terapêutico , Tomografia por Emissão de Pósitrons
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