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1.
Biomed Chromatogr ; 34(4): e4799, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31994209

RESUMO

Spermiogenesis in mammals is an exclusive process during which haploid round spermatids mature into spermatozoa in the testis. Any abnormality in the process of spermiogenesis may result in male infertility. The aim of the present study was to characterize the differentially expressed proteins between round and elongated spermatids in mice using label-free quantitative mass spectrometry. Of the 2411 proteins identified in this study, 333 were differentially expressed with a ≥10-fold change, including 208 upregulated proteins and 125 downregulated proteins in round spermatids relative to elongated spermatids. Gene Ontology analysis showed that these differentially expressed proteins were categorized into 10 types of subcellular localizations, 9 molecular functions, and were involved in 9 biological processes. All the identified proteins participated in 268 different pathways. In addition, ubiquitin-mediated proteolysis and the proteasome pathway, autophagy, lysosome, and apoptosis pathways were involved in the mechanism of spermiogenesis. Our data may provide valuable information for a better understanding of spermiogenesis and help improve the diagnosis and treatment of male factor infertility.


Assuntos
Proteoma/análise , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Bases de Dados de Proteínas , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos BALB C , Proteínas/análise , Proteínas/classificação , Proteínas/metabolismo , Proteoma/classificação , Proteoma/metabolismo , Espermátides/química
2.
Braz J Med Biol Res ; 53(1): e9001, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939598

RESUMO

Due to its various structures in bio-compounds, snake venom is the indisputable result of evolutionary stages of molecules with an increasingly complex structure, high specificity, and of great importance for medicine because of their potential. The present study proposed an underpinning examination of venom composition from nine species of venomous snakes using a useful and replicable methodology. The objective was the extension of the evaluation of protein fractions in the field up to 230 kDa to permit possible identification of some fractions that are insufficiently studied. The gel capillary electrophoresis method on the chip was performed using an Agilent 2100 bioassay with the 80 and 230-LabChip Protein kits. Interpretation of electrophoresis was performed using the Protein 2100 expert (Agilent) test software as follows: a) Protein 80 (peak size scale): 1.60, 3.5, 6.50, 15.00, 28.00, 46.00, 63.00, 95.00 kDa; b) Protein 230 (peak size scale): 4.50, 7.00, 15.00, 28.00, 46.00, 63.00, 95.00, 150.00, 240.00 kDa. The screening revealed the presence of compounds with a molecular weight greater than 80 kDa, in the case of Vipera aspis and Vipera xantina palestinae. For V. aspis, a 125 kDa molecular weight pro-coagulant protein was identified, known as being involved in the reduction of plasma clotting time without any direct activity in the fibrinogen coagulation process. The samples examined on the Protein 230-LabChip electrophoresis chip can be considered as a novelty with possible uses in medicine, requiring further approaches by advanced proteomics techniques to confirm the intimate structural features and biological properties of snake venoms.


Assuntos
Proteínas/química , Venenos de Víboras/química , Viperidae/classificação , Animais , Eletroforese Capilar , Proteínas/análise , Proteínas/isolamento & purificação , Proteoma/química , Proteoma/classificação , Proteômica/métodos , Venenos de Víboras/análise
3.
BMC Res Notes ; 12(1): 470, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370875

RESUMO

OBJECTIVE: Label-free quantitative proteomics has emerged as a powerful strategy to obtain high quality quantitative measures of the proteome with only a very small quantity of total protein extract. Because our research projects were requiring the application of bottom-up shotgun mass spectrometry proteomics in the pathogenic yeasts Candida glabrata and Candida albicans, we performed preliminary experiments to (i) obtain a precise list of all the proteins for which measures of abundance could be obtained and (ii) assess the reproducibility of the results arising respectively from biological and technical replicates. DATA DESCRIPTION: Three time-courses were performed in each Candida species, and an alkaline pH stress was induced for two of them. Cells were collected 10 and 60 min after stress induction and proteins were extracted. Samples were analysed two times by mass spectrometry. Our final dataset thus comprises label-free quantitative proteomics results for 24 samples (two species, three time-courses, two time points and two runs of mass spectrometry). Statistical procedures were applied to identify proteins with differential abundances between stressed and unstressed situations. Considering that C. glabrata and C. albicans are human pathogens, which face important pH fluctuations during a human host infection, this dataset has a potential value to other researchers in the field.


Assuntos
Candida albicans/genética , Candida glabrata/genética , Proteínas Fúngicas/genética , Proteoma/genética , Candida albicans/metabolismo , Candida glabrata/metabolismo , Conjuntos de Dados como Assunto , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Disseminação de Informação , Internet , Proteoma/classificação , Proteoma/metabolismo , Proteômica/métodos , Estresse Fisiológico/genética
4.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31331957

RESUMO

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes. Cyclophilins catalyze the cis-trans isomerization of xaa-proline bonds, a rate-limiting step in protein folding which has been shown to be important for bacterial virulence. B. pseudomallei carries a putative cyclophilin B gene, ppiB, the role of which was investigated. A B. pseudomallei ΔppiB (BpsΔppiB) mutant strain demonstrates impaired biofilm formation and reduced motility. Macrophage invasion and survival assays showed that although the BpsΔppiB strain retained the ability to infect macrophages, it had reduced survival and lacked the ability to spread cell to cell, indicating ppiB is essential for B. pseudomallei virulence. This is reflected in the BALB/c mouse infection model, demonstrating the requirement of ppiB for in vivo disease dissemination and progression. Proteomic analysis demonstrates that the loss of PpiB leads to pleiotropic effects, supporting the role of PpiB in maintaining proteome homeostasis. The loss of PpiB leads to decreased abundance of multiple virulence determinants, including flagellar machinery and alterations in type VI secretion system proteins. In addition, the loss of ppiB leads to increased sensitivity toward multiple antibiotics, including meropenem and doxycycline, highlighting ppiB inhibition as a promising antivirulence target to both treat B. pseudomallei infections and increase antibiotic efficacy.


Assuntos
Proteínas de Bactérias/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidade , Ciclofilinas/genética , Melioidose/microbiologia , Proteoma/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/efeitos dos fármacos , Burkholderia pseudomallei/metabolismo , Linhagem Celular , Ciclofilinas/deficiência , Feminino , Deleção de Genes , Expressão Gênica , Homeostase/genética , Macrófagos/microbiologia , Melioidose/tratamento farmacológico , Melioidose/mortalidade , Melioidose/patologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/efeitos dos fármacos , Proteoma/classificação , Proteoma/metabolismo , Análise de Sobrevida , Virulência
5.
Mol Cell ; 75(1): 184-199.e10, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31076284

RESUMO

The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , Mapas de Interação de Proteínas , Proteoma/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Fatores de Transcrição/genética , Centrifugação com Gradiente de Concentração/instrumentação , Cromatina/química , Cromatina/metabolismo , Regulação da Expressão Gênica , Ontologia Genética , Células HeLa , Humanos , Disseminação de Informação , Internet , Anotação de Sequência Molecular , Ligação Proteica , Proteoma/classificação , Proteoma/metabolismo , Proteômica/métodos , RNA/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo
6.
Molecules ; 24(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018578

RESUMO

Legume crops represent the major source of food protein and contribute to human nutrition and animal feeding. An essential improvement of their productivity can be achieved by symbiosis with beneficial soil microorganisms-rhizobia (Rh) and arbuscular mycorrhizal (AM) fungi. The efficiency of these interactions depends on plant genotype. Recently, we have shown that, after simultaneous inoculation with Rh and AM, the productivity gain of pea (Pisum sativum L) line K-8274, characterized by high efficiency of interaction with soil microorganisms (EIBSM), was higher in comparison to a low-EIBSM line K-3358. However, the molecular mechanisms behind this effect are still uncharacterized. Therefore, here, we address the alterations in pea seed proteome, underlying the symbiosis-related productivity gain, and identify 111 differentially expressed proteins in the two lines. The high-EIBSM line K-8274 responded to inoculation by prolongation of seed maturation, manifested by up-regulation of proteins involved in cellular respiration, protein biosynthesis, and down-regulation of late-embryogenesis abundant (LEA) proteins. In contrast, the low-EIBSM line K-3358 demonstrated lower levels of the proteins, related to cell metabolism. Thus, we propose that the EIBSM trait is linked to prolongation of seed filling that needs to be taken into account in pulse crop breeding programs. The raw data have been deposited to the ProteomeXchange with identifier PXD013479.


Assuntos
Regulação da Expressão Gênica de Plantas , Ervilhas/genética , Proteínas de Plantas/isolamento & purificação , Proteoma/isolamento & purificação , Sementes/genética , Simbiose/genética , Bactérias/crescimento & desenvolvimento , Biomassa , Cromatografia Líquida de Alta Pressão , Fungos/fisiologia , Ontologia Genética , Genótipo , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Micorrizas/fisiologia , Ervilhas/química , Ervilhas/metabolismo , Ervilhas/microbiologia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Nodulação/genética , Proteoma/classificação , Proteoma/genética , Proteômica/métodos , Sementes/química , Sementes/metabolismo , Microbiologia do Solo , Espectrometria de Massas em Tandem
7.
Nat Commun ; 10(1): 1311, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30899004

RESUMO

Protein glycosylation is a highly important, yet poorly understood protein post-translational modification. Thousands of possible glycan structures and compositions create potential for tremendous site heterogeneity. A lack of suitable analytical methods for large-scale analyses of intact glycopeptides has limited our abilities both to address the degree of heterogeneity across the glycoproteome and to understand how this contributes biologically to complex systems. Here we show that N-glycoproteome site-specific microheterogeneity can be captured via large-scale glycopeptide profiling methods enabled by activated ion electron transfer dissociation (AI-ETD), ultimately characterizing 1,545 N-glycosites (>5,600 unique N-glycopeptides) from mouse brain tissue. Our data reveal that N-glycosylation profiles can differ between subcellular regions and structural domains and that N-glycosite heterogeneity manifests in several different forms, including dramatic differences in glycosites on the same protein. Moreover, we use this large-scale glycoproteomic dataset to develop several visualizations that will prove useful for analyzing intact glycopeptides in future studies.


Assuntos
Encéfalo/metabolismo , Glicopeptídeos/química , Proteínas do Tecido Nervoso/química , Polissacarídeos/química , Processamento de Proteína Pós-Traducional , Proteoma/química , Animais , Química Encefálica , Conjuntos de Dados como Assunto , Feminino , Expressão Gênica , Glicopeptídeos/classificação , Glicopeptídeos/genética , Glicopeptídeos/isolamento & purificação , Glicosilação , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Polissacarídeos/isolamento & purificação , Proteoma/classificação , Proteoma/genética , Proteoma/isolamento & purificação , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
8.
PLoS Biol ; 17(2): e3000154, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30794532

RESUMO

Cyclic nucleotide signalling is a major regulator of malaria parasite differentiation. Phosphodiesterase (PDE) enzymes are known to control cyclic GMP (cGMP) levels in the parasite, but the mechanisms by which cyclic AMP (cAMP) is regulated remain enigmatic. Here, we demonstrate that Plasmodium falciparum phosphodiesterase ß (PDEß) hydrolyses both cAMP and cGMP and is essential for blood stage viability. Conditional gene disruption causes a profound reduction in invasion of erythrocytes and rapid death of those merozoites that invade. We show that this dual phenotype results from elevated cAMP levels and hyperactivation of the cAMP-dependent protein kinase (PKA). Phosphoproteomic analysis of PDEß-null parasites reveals a >2-fold increase in phosphorylation at over 200 phosphosites, more than half of which conform to a PKA substrate consensus sequence. We conclude that PDEß plays a critical role in governing correct temporal activation of PKA required for erythrocyte invasion, whilst suppressing untimely PKA activation during early intra-erythrocytic development.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Transdução de Sinais/genética , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Eritrócitos/parasitologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hidrólise , Merozoítos/enzimologia , Merozoítos/genética , Merozoítos/crescimento & desenvolvimento , Fosfoproteínas/classificação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteoma/classificação , Proteoma/genética , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Esquizontes/enzimologia , Esquizontes/genética , Esquizontes/crescimento & desenvolvimento , Fatores de Tempo
9.
Mar Biotechnol (NY) ; 21(1): 38-51, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30413912

RESUMO

Adhesion in barnacles is still poorly understood. The cement gland secretes an insoluble multi-protein complex, which adheres very strongly to a variety of substrates in the presence of water. This adhesion mechanism is bioinspiring for the engineering of new adhesive materials, but to replicate this adhesive system, the genes coding for the cement constitutive proteins must be identified and elucidated, and their products characterised. Here, the complete sequences of three cement protein (CP) genes (CP-100K, CP-52K, and CP-19K) isolated from the cement gland of the stalked barnacle Pollicipes pollicipes (order Scalpelliformes) were obtained using RACE PCR. The three genes were compared to the 23 other acorn barnacle CP genes so far sequenced (order Sessilia) to determine common and differential patterns and molecular properties, since the adhesives of both orders have visibly different characteristics. A shotgun proteomic analysis was performed on the cement, excreted at the membranous base of specimens, where the products of the three genes sequenced in the gland were identified, validating their function as CPs. A principal component analysis (PCA) was performed, to cluster CPs into groups with similar amino acid composition. This analysis uncovered three CP groups, each characterised by similar residue composition, features in secondary structure, and some biochemical properties, including isoelectric point and residue accessibility to solvents. The similarity among proteins in each defined group was low despite comparable amino acid composition. PCA can identify putative adhesive proteins from NGS transcriptomic data regardless of their low homology. This analysis did not highlight significant differences in residue composition between homologous acorn and stalked barnacle CPs. The characteristics responsible for the structural differences between the cement of stalked and acorn barnacles are described, and the presence of nanostructures, such as repetitive homologous domains and low complexity regions, and repetitive ß-sheets are discussed relatively to self-assembly and adhesion.


Assuntos
Adesivos/química , Proteínas de Artrópodes/química , Proteoma/química , Thoracica/química , Adesivos/classificação , Adesivos/metabolismo , Sequência de Aminoácidos , Animais , Organismos Aquáticos , Proteínas de Artrópodes/classificação , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Ponto Isoelétrico , Anotação de Sequência Molecular , Análise de Componente Principal , Proteoma/classificação , Proteoma/genética , Proteoma/metabolismo , Alinhamento de Sequência , Thoracica/genética , Thoracica/metabolismo , Transcriptoma
10.
Mar Biotechnol (NY) ; 21(1): 99-110, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30456696

RESUMO

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.


Assuntos
Proteínas de Algas/genética , Meios de Cultura/farmacologia , Ácidos Docosa-Hexaenoicos/biossíntese , Ácido Eicosapentaenoico/biossíntese , Proteoma/genética , Estramenópilas/efeitos dos fármacos , Proteínas de Algas/classificação , Proteínas de Algas/isolamento & purificação , Biomassa , Carotenoides/biossíntese , Carotenoides/isolamento & purificação , Meios de Cultura/química , Ácidos Docosa-Hexaenoicos/isolamento & purificação , Ácido Eicosapentaenoico/isolamento & purificação , Expressão Gênica , Ontologia Genética , Glucose/metabolismo , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Maltose/metabolismo , Maltose/farmacologia , Anotação de Sequência Molecular , Proteoma/classificação , Proteoma/isolamento & purificação , RNA Ribossômico 18S/genética , Água do Mar/química , Análise de Sequência de DNA , Amido/metabolismo , Amido/farmacologia , Estramenópilas/genética , Estramenópilas/crescimento & desenvolvimento , Estramenópilas/metabolismo
11.
Cell ; 175(4): 1141-1155.e16, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30343902

RESUMO

CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.


Assuntos
Sistemas CRISPR-Cas , Citometria de Fluxo/métodos , Genômica/métodos , Espectrometria de Massas/métodos , Análise de Célula Única/métodos , Animais , Epitopos/química , Epitopos/classificação , Epitopos/genética , Células HEK293 , Humanos , Imunofenotipagem/métodos , Células Jurkat , Camundongos Endogâmicos BALB C , Proteoma/química , Proteoma/classificação , Proteoma/genética , Células THP-1
12.
J Proteome Res ; 17(11): 3904-3913, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30223649

RESUMO

Jellyfish are a type of poisonous cnidarian invertebrate that secrete lethal venom for predation or defense. Human beings often become victims of jellyfish stings accidentally while swimming or fishing and suffer severe pain, itching, swelling, inflammation, shock, and even death. Jellyfish venom is composed of various toxins, and the lethal toxin is the most toxic and hazardous component of the venom, which is responsible for deaths caused by jellyfish stings and envenomation. Our previous study revealed many toxins in jellyfish venom, including phospholipase A2, metalloproteinase, and protease inhibitors. However, it is still unknown which type of toxin is lethal and how it works. Herein a combined toxicology analysis, proteome strategy, and purification approach was employed to investigate the lethality of the venom of the jellyfish Cyanea nozakii. Toxicity analysis revealed that cardiotoxicity including acute myocardial infarction and a significant decrease in both heart rate and blood pressure is the primary cause of death. Purified lethal toxin containing a fraction of jellyfish venom was subsequently subjected to proteome analysis and bioinformation analysis. A total of 316 and 374 homologous proteins were identified, including phospholipase A2-like toxins and metalloprotease-like toxins. Furthermore, we confirmed that the lethality of the jellyfish venom is related to metalloproteinase activity but without any phospholipase A2 activity or hemolytic activity. Altogether, this study not only provides a comprehensive understanding of the lethal mechanism of jellyfish venom but also provides very useful information for the therapeutic or rescue strategy for severe jellyfish stings.


Assuntos
Venenos de Cnidários/química , Metaloproteases/isolamento & purificação , Infarto do Miocárdio/induzido quimicamente , Fosfolipases A2/isolamento & purificação , Proteoma/isolamento & purificação , Cifozoários/química , Animais , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Cromatografia Líquida , Venenos de Cnidários/toxicidade , Feminino , Ontologia Genética , Coração/efeitos dos fármacos , Coração/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Masculino , Espectrometria de Massas , Metaloproteases/química , Metaloproteases/toxicidade , Camundongos , Anotação de Sequência Molecular , Infarto do Miocárdio/fisiopatologia , Fosfolipases A2/química , Fosfolipases A2/toxicidade , Proteoma/química , Proteoma/classificação , Proteoma/toxicidade , Proteômica/métodos , Cifozoários/patogenicidade , Cifozoários/fisiologia , Baço/efeitos dos fármacos , Baço/fisiopatologia
13.
J Proteome Res ; 17(11): 3761-3773, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30261726

RESUMO

Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin-magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented N-glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/química , Glicopeptídeos/isolamento & purificação , Glicoproteínas/isolamento & purificação , Lectinas/química , Neoplasias Pulmonares/química , Proteoma/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Sequência de Carboidratos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Glicoconjugados/química , Glicoconjugados/metabolismo , Glicopeptídeos/classificação , Glicopeptídeos/genética , Glicopeptídeos/metabolismo , Glicoproteínas/classificação , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Humanos , Lectinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Nanopartículas de Magnetita/química , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Proteoma/classificação , Proteoma/genética , Proteoma/metabolismo , Proteômica
14.
J Proteome Res ; 17(9): 3128-3142, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30079738

RESUMO

Aflatoxin M1 (AFM1) is a common mycotoxin in dairy milk, and it is typically concurrently present with other mycotoxins that may represent a threat to food safety. However, knowledge of how AFM1, alone or in combination with other mycotoxins, may affect human intestinal epithelial integrity remain to be established. We employed transcriptome and proteome analysis integrated with biological validation to reveal the molecular basis underlining the effect of exposure to AFM1, ochratoxin A (OTA), or both on the intestinal epithelial integrity of differentiated Caco-2 cells. Exposure to 4 µg/mL of OTA was found to disrupt human gut epithelial integrity, whereas 4 µg/mL of AFM1 did not. The integrated transcriptome and proteome analysis of AFM1 and OTA, alone or in combination, indicate the synergistic effect of the two mycotoxins in disrupting intestinal integrity. This effect was mechanistically linked to a broad range of pathways related to intestinal integrity enriched by down-regulated genes and proteins, associated with focal adhesion, adheren junction, and gap junction pathways. Furthermore, the cross-omics analysis of mixed AFM1 and OTA compared to OTA alone suggest that kinase family members, including myosin light-chain kinase, mitogen-activated protein kinases, and protein kinase C, are the potential key regulators in modulating intestinal epithelial integrity. These findings provide novel insight into the synergistic detrimental role of multiple mycotoxins in disrupting intestinal integrity and, therefore, identify potential targets to improve milk safety related to human health.


Assuntos
Aflatoxina M1/toxicidade , Adesões Focais/efeitos dos fármacos , Ocratoxinas/toxicidade , Proteoma/genética , Transcriptoma , Junções Aderentes/efeitos dos fármacos , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Junções Comunicantes/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Mapas de Interação de Proteínas , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteoma/classificação , Proteoma/metabolismo
15.
J Proteome Res ; 17(9): 3308-3316, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30080416

RESUMO

Analysis of protein phosphorylation in extracellular vesicles (EVs) offers an unprecedented potential for understanding cancer signaling and early stage disease diagnosis. However, prior to the phosphoproteome analysis step, the isolation of EVs from biofluids remains a challenging issue to overcome due to the low yield and impurity from current isolation methods. Here, we carry out an extensive assessment of several EV isolation methods including a novel rapid isolation method EVTRAP for highly efficient capture of extracellular vesicles from human urine sample. We demonstrate that over 95% recovery yield can be consistently achieved by EVTRAP, a significant improvement over current standard techniques. We then applied EVTRAP to identify over 16 000 unique peptides representing 2000 unique EV proteins from 200 µL urine sample, including all known EV markers with substantially increased recovery levels over ultracentrifugation. Most importantly, close to 2000 unique phosphopeptides were identified from more than 860 unique phosphoproteins using 10 mL of urine. The data demonstrated that EVTRAP is a highly effective and potentially widely implementable clinical isolation method for analysis of EV protein phosphorylation.


Assuntos
Técnicas de Química Analítica/instrumentação , Vesículas Extracelulares/química , Fosfopeptídeos/análise , Fosfoproteínas/isolamento & purificação , Proteoma/isolamento & purificação , Biomarcadores/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imãs , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/classificação , Fosfoproteínas/urina , Ligação Proteica , Proteoma/química , Proteoma/classificação , Tetraspanina 29/química , Tetraspanina 29/metabolismo , Ultracentrifugação
16.
J Proteome Res ; 17(9): 2995-3011, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30106293

RESUMO

Huanglongbing (HLB), also known as citrus greening disease, is the most serious disease of citrus plants. It is associated with the Gram-negative bacterium ' Candidatus Liberibacter asiaticus' ( CLas), which is transmitted between host plants by the hemipteran insect vector Diaphorina citri in a circulative, propagative manner involving specific interactions with various insect tissues including the hemolymph, fluid that occupies the body cavity akin to insect blood. High resolution quantitative mass spectrometry was performed to investigate the effect of CLas exposure on D. citri hemolymph at the proteome level. In contrast to the broad proteome effects on hundreds of proteins and a diverse array of metabolic pathways previously reported in gut and whole insect proteome analyses, the effect of CLas on the hemolymph was observed to be highly specific, restricted to key immunity and metabolism pathways, and lower in magnitude than that previously observed in the whole insect body and gut. Vitellogenins were abundantly expressed and CLas-responsive. Gene-specific RNA expression analysis suggests that these proteins are expressed in both male and female insects and may have roles outside of reproductive vitellogenesis. Proteins for fatty acid synthesis were found to be up-regulated, along with metabolic proteins associated with energy production, supported at the organismal level by the previously published observation that D. citri individuals experience a higher level of hunger when reared on CLas-infected plants. Prediction of post-translational modifications identified hemolymph proteins with phosphorylation and acetylation upon CLas exposure. Proteins derived from the three most prominent bacterial endosymbionts of the psyllid were also detected in the hemolymph, and several of these have predicted secretion signals. A DNAK protein, the bacterial HSP70, detected in the hemolymph expressed from Wolbachia pipientis was predicted to encode a eukaryotic nuclear localization signal. Taken together, these data show specific changes to immunity and metabolism in D. citri hemolymph involving host and endosymbiont proteins. These data provide a novel context for proteomic changes seen in other D. citri tissues in response to CLas and align with organismal data on the effects of CLas on D. citri metabolism and reproduction.


Assuntos
Proteínas de Bactérias/metabolismo , Hemípteros/metabolismo , Hemolinfa/química , Proteínas de Insetos/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Rhizobiaceae/metabolismo , Acetilação , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Citrus/parasitologia , Metabolismo Energético , Ácidos Graxos , Ontologia Genética , Hemípteros/genética , Hemípteros/imunologia , Hemípteros/microbiologia , Hemolinfa/imunologia , Hemolinfa/metabolismo , Hemolinfa/microbiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Insetos Vetores/genética , Insetos Vetores/imunologia , Insetos Vetores/metabolismo , Insetos Vetores/microbiologia , Metabolismo dos Lipídeos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Anotação de Sequência Molecular , Fosforilação , Doenças das Plantas/parasitologia , Proteoma/classificação , Proteoma/genética , Proteoma/imunologia , Proteômica/métodos , Rhizobiaceae/genética , Simbiose/genética , Simbiose/imunologia , Vitelogeninas , Wolbachia/genética , Wolbachia/metabolismo
17.
J Proteome Res ; 17(9): 3153-3175, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30111112

RESUMO

Periodontitis is a prevalent chronic inflammatory disease associated with dysbiosis. Although complement inhibition has been successfully used to treat periodontitis in animal models, studies globally analyzing inflamed tissue proteins to glean insight into possible mechanisms of action are missing. Using quantitative shotgun proteomics, we aimed to investigate differences in composition of inflammatory gingival tissue exudate ("gingival crevicular fluid"; GCF), before and after local administration of an inhibitor of the central complement component, C3, in nonhuman primates. The C3 inhibitor, Cp40 (also known as AMY-101) was administered locally in the maxillary gingival tissue of cynomolgus monkeys with established periodontitis, either once a week (1×-treatment; n = 5 animals) or three times per week (3×-treatment; n = 10 animals), for 6 weeks followed by another 6 weeks of observation in the absence of treatment. 45 GCF samples were processed for FASP digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Data were processed using the ProgenesisQI software. The statistical significance of differences between the groups was determined by RM-ANOVA, and a protein expression change was considered as a true regulation at >2-fold and p < 0.05. The human orthologues were subjected to Gene Ontology analyses using PANTHER. Data are available via ProteomeXchange with identifier PXD009502. 573 proteins with >2 peptides were longitudinally quantified. Both 3× and 1× administration of Cp40 resulted in significant down-regulation of dozens of proteins during the 6-week course of treatment as compared to baseline. Following drug withdrawal at 6 weeks, more than 50% of the down-regulated proteins showed increased levels at week 12. The top scored pathway was "complement activation, alternative pathway", and several proteins involved in this pathway were down-regulated at 6 weeks. We mapped the proteomic fingerprint changes in local tissue exudate of cynomolgus monkey periodontitis in response to C3 inhibition and identified the alternative pathway of complement activation and leukocyte degranulation as main targets, which are thus likely to play significant roles in periodontal disease pathogenesis. Label-free quantitative proteomics strategies utilizing GCF are powerful tools for the identification of treatment targets and providing insights into disease mechanisms.


Assuntos
Anti-Inflamatórios/farmacologia , Complemento C3/antagonistas & inibidores , Via Alternativa do Complemento/efeitos dos fármacos , Líquido do Sulco Gengival/química , Peptídeos Cíclicos/farmacologia , Periodontite/tratamento farmacológico , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Cromatografia Líquida , Complemento C3/genética , Via Alternativa do Complemento/genética , Modelos Animais de Doenças , Esquema de Medicação , Regulação da Expressão Gênica , Ontologia Genética , Gengiva/efeitos dos fármacos , Gengiva/imunologia , Gengiva/patologia , Líquido do Sulco Gengival/efeitos dos fármacos , Líquido do Sulco Gengival/imunologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/patologia , Macaca fascicularis , Anotação de Sequência Molecular , Periodontite/genética , Periodontite/imunologia , Periodontite/patologia , Proteoma/classificação , Proteoma/genética , Proteoma/imunologia , Espectrometria de Massas em Tandem
18.
Proteomics Clin Appl ; 12(6): e1800041, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30054970

RESUMO

PURPOSE: Approximately 5% of giant cell tumors (GCT) of bone develop pulmonary metastases. Although many biomarkers have been proposed, identification of circulating low abundance molecules may be useful to predict malignant progression. EXPERIMENTAL DESIGN: The hydrogel nanoparticle technique followed by MS was used to detect low molecular weight serum proteins or protein fragments in serum of 20 GCT patients with different clinical course and in ten healthy sera used as control. The most representative low-abundant de novo or differentially abundant proteins were submitted to String database that recognized interconnected activated pathways including protein activation cascade, wound healing, cell-substrate adhesion, and response to stress. Statistics were performed for identification of candidate prognostic factors. RESULTS: Proteome cluster analysis separated metastasis-free from metastatic GCT patients in two well-defined groups where serum levels of signaling transduction mediators and regulators of kinase activity presented a high discriminatory power. Increased expression of proteins STAT5B, GRB2, and OXSR1 was related to a higher probability of metastasis. Multivariate analysis demonstrated that tumor grade and STAT5B were independent prognostic factors. CONCLUSIONS AND CLINICAL RELEVANCE: By using a noninvasive technique, we identified differentially abundant serum candidate biomarkers, also providing prognostic information in patients with GCT of bone.


Assuntos
Neoplasias Ósseas/sangue , Proteína Adaptadora GRB2/sangue , Tumores de Células Gigantes/sangue , Neoplasias Pulmonares/sangue , Proteínas Serina-Treonina Quinases/sangue , Fator de Transcrição STAT5/sangue , Adolescente , Adulto , Biomarcadores Tumorais/sangue , Neoplasias Ósseas/epidemiologia , Neoplasias Ósseas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Tumores de Células Gigantes/epidemiologia , Tumores de Células Gigantes/patologia , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Pessoa de Meia-Idade , Nanopartículas/química , Gradação de Tumores , Metástase Neoplásica , Células Neoplásicas Circulantes , Prognóstico , Proteoma/classificação , Proteoma/genética , Fatores de Risco , Adulto Jovem
19.
Int J Mol Sci ; 19(7)2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-30011845

RESUMO

Human adipose-derived mesenchymal stem cells (hADSCs) are representative cell sources for cell therapy. Classically, Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) has been used as culture medium for hADSCs. A chemically defined medium (CDM) containing no heterologous animal components has recently been used to produce therapeutic hADSCs. However, how the culture environment using a medium without FBS affects the protein expression of hADSC is unclear. We subjected hADSCs cultured in CDM and DMEM (10% FBS) to a protein expression analysis by tandem mass spectrometry liquid chromatography and noted 98.2% agreement in the proteins expressed by the CDM and DMEM groups. We classified 761 proteins expressed in both groups by their function in a gene ontology analysis. Thirty-one groups of proteins were classified as growth-related proteins in the CDM and DMEM groups, 16 were classified as antioxidant activity-related, 147 were classified as immune system process-related, 557 were involved in biological regulation, 493 were classified as metabolic process-related, and 407 were classified as related to stimulus responses. These results show that the trend in the expression of major proteins related to the therapeutic effect of hADSCs correlated strongly in both groups.


Assuntos
Cromatografia Líquida/métodos , Células-Tronco Mesenquimais/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Tecido Adiposo/citologia , Animais , Bovinos , Técnicas de Cultura de Células , Células Cultivadas , Análise por Conglomerados , Meios de Cultura/química , Meios de Cultura/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteoma/classificação , Soro/química
20.
Protein Expr Purif ; 152: 13-22, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30017744

RESUMO

A proteomic approach was used to understand the molecular mechanisms underlying obstacles to the continuous cropping of Pogostemon cablin. We examined differences in protein abundance between control (CK) and continuously cropped (TR) P. cablin leaves at different time points (90, 150, and 210 days after culture). Comparative analysis by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) revealed 183 differentially expressed protein spots, of which 87 proteins or isoforms were identified using mass spectrometry. Among these differentially expressed proteins (DEPs), 50 proteins or isoforms showed increased abundance and 37 proteins or isoforms showed decreased abundance in the TR sample compared with the abundance in the CK sample. Bioinformatic tools were used to analyze the DEPs. These proteins were classified into 12 categories according to clusters of orthologous groups (COG) analysis, with the majority being involved in post-translational modification, protein turnover, and chaperones, followed by carbohydrate transport and metabolism, and finally, energy production and conversion. Protein-protein interactions revealed that 18 DEPs were involved in energy metabolism, 6 DEPs were involved in stress response, and 4 DEPs were involved in amino acid biosynthesis. Continuous cropping altered the expression of proteins related to energy metabolism, carbohydrate metabolism, and amino acid metabolism in P. cablin leaves. Among these processes, the most affected was energy metabolism, which may be pivotal for resistance to continuous cropping.


Assuntos
Regulação da Expressão Gênica de Plantas , Folhas de Planta/genética , Proteínas de Plantas/genética , Pogostemon/genética , Processamento de Proteína Pós-Traducional , Proteoma/genética , Transporte Biológico/genética , Metabolismo dos Carboidratos/genética , Biologia Computacional/métodos , Metabolismo Energético/genética , Ontologia Genética , Chaperonas Moleculares/classificação , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Anotação de Sequência Molecular , Folhas de Planta/química , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Pogostemon/química , Pogostemon/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/classificação , Proteoma/metabolismo
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