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1.
Arch Virol ; 164(12): 2995-3006, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31576460

RESUMO

Reticuloendotheliosis virus (REV) is an important representative avian retrovirus. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of REV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect changes in protein levels in chicken embryo fibroblast cells (CEFs) that were infected with REV or mock infected. In total, 605 cellular proteins were differentially expressed, among which 196, 345, and 286 were differentially expressed in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and immune system processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding, catalytic activity, and enzyme regulator activity. Pathway analysis showed that a total of 143, 167, and 179 pathways, including protein digestion and absorption, focal adhesion, ECM-receptor interaction, cytokine-cytokine receptor interaction, Toll-like receptors, and JAK-STAT signaling, were enriched in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. In conclusion, this study is the first to analyze the protein profile of REV-infected CEFs using an iTRAQ approach. The results of this study provide valuable information for better understanding the host response to REV infection.


Assuntos
Fibroblastos/metabolismo , Doenças das Aves Domésticas/genética , Proteoma/genética , Vírus da Reticuloendoteliose/fisiologia , Infecções por Retroviridae/veterinária , Animais , Embrião de Galinha , Galinhas , Fibroblastos/química , Fibroblastos/virologia , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/virologia , Proteoma/química , Proteoma/metabolismo , Proteômica , Vírus da Reticuloendoteliose/genética , Infecções por Retroviridae/genética , Infecções por Retroviridae/metabolismo , Infecções por Retroviridae/virologia , Espectrometria de Massas em Tandem
2.
Mol Biol (Mosk) ; 53(4): 685-691, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397442

RESUMO

In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Proteoma/análise , Proteômica/métodos , Coloração e Rotulagem/métodos , Humanos , Focalização Isoelétrica , Proteoma/química , Espectrometria de Massas em Tandem
3.
Chemistry ; 25(54): 12644-12651, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31310394

RESUMO

The illudin natural product family are fungal secondary metabolites with a characteristic spirocyclopropyl-substituted fused 6,5-bicyclic ring system. They have been extensively studied for their cytotoxicity in various tumor cell types, and semisynthetic derivatives with improved therapeutic characteristics have progressed to clinical trials. Although it is believed that this potent alkylating compound class acts mainly through DNA modification, little is known about its binding to protein sites in a cellular context. To reveal putative protein targets of the illudin family in live cancer cells, we employed a semisynthetic strategy to access a series of illudin-based probes for activity-based protein profiling (ABPP). While the probes largely retained potent cytotoxicity, proteomic profiling studies unraveled multiple protein hits, suggesting that illudins exert their mode of action not from addressing a specific protein target but rather from DNA modification and unselective protein binding.


Assuntos
Proteínas/química , Proteoma/química , Sesquiterpenos/farmacologia , Compostos de Espiro/farmacologia , Células A549 , Alquilação , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Sobrevivência Celular , Humanos , Ligação Proteica , Proteínas/metabolismo , Proteoma/metabolismo , Metabolismo Secundário , Sesquiterpenos/química , Compostos de Espiro/química
4.
Anal Chim Acta ; 1076: 82-90, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203967

RESUMO

We present a method to preserve and process urine proteins for proteomic analysis in a filter aided sample preparation (FASP) format. The method combines concentration of urine proteins in ultrafiltration devices, their thermal stabilization, allowing long term storage of the samples, and filter aided sample preparation. Proteomes of four different urines were preserved during 48 h and 6 months using (i) the classic freeze preservation at -20 °C, (ii) snap-heated freeze-free preservation at laboratory temperature (20 °C) and (iii) snap-heated preservation at -60 °C. The three storage methods can effetely preserve the urine proteome for at least 6 months without significant alterations. Abundances of more than 500 proteins and specially 24 selected -cleared or -approved protein assayed in serum or plasma were found similar within the three preservation methods assessed. The new method here proposed dramatically simplifies the conditions for preserving the urine proteome for biobanks in terms of space and storage, including lowering the risks of sample degradation caused by misfunction of the freezer. Furthermore, the shipping of large number of samples can be made without the need of freezing. The application of the FASP format to isolate and preserve the proteins facilitates long-term storage and processing of proteome of urine samples.


Assuntos
Proteoma/química , Manejo de Espécimes/métodos , Urina/química , Lesão Renal Aguda/urina , Adulto , Biomarcadores/análise , Análise por Conglomerados , Calefação , Humanos , Biópsia Líquida , Masculino , Estudo de Prova de Conceito , Proteoma/análise , Proteoma/isolamento & purificação , Adulto Jovem
5.
Mol Biol (Mosk) ; 53(3): 524-528, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184618

RESUMO

Proteins with homo-repeats of more than 4 amino acid residues in length were examined to understand whether some splicing sites in pre-mRNA may be attributed to homo-repeats in human proteins. The human proteome was found to contain a total of 404 proteins with homo-repeats that account for at least one splicing site in pre-mRNA. Pre-mRNA splicing sites were more often found in the C-terminal part (67%) than in the middle orN-terminal part of a homo-repeat. Ten homo-repeats were identified to have two splicing sites per repeat. The repeats were lysine homo-repeats in all but one case.


Assuntos
Proteínas/análise , Proteínas/química , Precursores de RNA/genética , Sítios de Splice de RNA/genética , Sequências Repetitivas de Aminoácidos/genética , Humanos , Lisina/genética , Lisina/metabolismo , Proteínas/genética , Proteoma/análise , Proteoma/química , Proteoma/genética , Processamento de RNA/genética
6.
Int J Mol Sci ; 20(9)2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31052352

RESUMO

Sarcoidosis is a systemic interstitial lung disease of unknown aetiology. Less invasive diagnostics are needed to decipher disease pathology and to distinguish sub-phenotypes. Here we test if SpotLight proteomics, which combines de novo MS/MS sequencing of enriched IgG and co-extracted proteins with subsequent label-free quantification of new and known peptides, can differentiate controls and sarcoidosis phenotypes (Löfgrens and non-Löfgrens syndrome, LS and nonLS). Intra-individually matched IgG enriched from serum and bronchial lavage fluid (BALF) from controls (n = 12), LS (n = 11) and nonLS (n = 12) were investigated. High-resolution mass-spectrometry SpotLight proteomics and uni- and multivariate-statistical analyses were used for data processing. Major differences were particularly observed in control-BALF versus sarcoidosis-BALF. However, interestingly, information obtained from BALF profiles was still present (but less prominent) in matched serum profiles. By using information from orthogonal partial least squares discriminant analysis (OPLS-DA) differentiating 1) sarcoidosis-BALF and control-BALF and 2) LS-BALF vs. nonLS-BALF, control-serum and sarcoidosis-serum (p = 0.0007) as well as LS-serum and nonLS-serum (p = 0.006) could be distinguished. Noteworthy, many factors prominent in identifying controls and patients were those associated with Fc-regulation, but also features from the IgG-Fab region and novel peptide variants. Differences between phenotypes were mostly IgG-specificity related. The results support the analytical utility of SpotLight proteomics which prospectively have potential to differentiate closely related phenotypes from a simple blood test.


Assuntos
Imunoglobulina G/sangue , Proteoma/química , Sarcoidose/sangue , Adulto , Biomarcadores/análise , Biomarcadores/sangue , Líquido da Lavagem Broncoalveolar/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Proteoma/metabolismo , Sarcoidose/metabolismo
7.
PLoS Negl Trop Dis ; 13(5): e0007362, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31091291

RESUMO

BACKGROUND: Schistosomiasis is a neglected disease affecting hundreds of millions worldwide. Of the three main species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. S. haematobium infection can cause different urogenital clinical complications, particularly in the bladder, and furthermore, this parasite has been strongly linked with squamous cell carcinoma. A comprehensive analysis of the molecular composition of its different proteomes will contribute to developing new tools against this devastating disease. METHODS AND FINDINGS: By combining a comprehensive protein fractionation approach consisting of OFFGEL electrophoresis with high-throughput mass spectrometry, we have performed the first in-depth characterisation of the different discrete proteomes of S. haematobium that are predicted to interact with human host tissues, including the secreted and tegumental proteomes of adult flukes and secreted and soluble egg proteomes. A total of 662, 239, 210 and 138 proteins were found in the adult tegument, adult secreted, soluble egg and secreted egg proteomes, respectively. In addition, we probed these distinct proteomes with urine to assess urinary antibody responses from naturally infected human subjects with different infection intensities, and identified adult fluke secreted and tegument extracts as being the best predictors of infection. CONCLUSION: We provide a comprehensive dataset of proteins from the adult and egg stages of S. haematobium and highlight their utility as diagnostic markers of infection intensity. Protein composition was markedly different between the different extracts, highlighting the distinct subsets of proteins that different development stages present in their different niches. Furthermore, we have identified adult fluke ES and tegument extracts as best predictors of infection using urine antibodies of naturally infected people. This study provides the first steps towards the development of novel tools to control this important neglected tropical disease.


Assuntos
Proteínas de Helminto/metabolismo , Proteoma/metabolismo , Schistosoma haematobium/metabolismo , Esquistossomose Urinária/parasitologia , Animais , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Masculino , Proteoma/química , Proteoma/genética , Proteômica , Schistosoma haematobium/química , Schistosoma haematobium/classificação , Schistosoma haematobium/genética
8.
Nat Chem ; 11(6): 552-561, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936521

RESUMO

Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.


Assuntos
Alquinos/química , Sondas Moleculares/química , Prenilação de Proteína , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Espectrometria de Massas , Camundongos Knockout , Prenilação de Proteína/efeitos dos fármacos , Proteínas/química , Proteoma/química
9.
Theriogenology ; 132: 53-61, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30991169

RESUMO

The oviduct provides the optimal micro milieu for early embryo development. However, accessing the bovine oviductal fluid in vivo for analysis is still challenging and therefore the oviductal fluid is usually collected post mortem. In the study presented here we introduce a novel approach to gain minimal invasive access to the bovine oviductal fluid proteome in vivo by transvaginal endoscopy at different stages of the estrous cycle. The first experiment aimed at transferring C4 derivatised magnetic beads to bind the oviductal fluid proteome in situ. Protein carrying beads were recovered by flushing the oviduct and proteins were eluted. In the second experiment a flushing solution was injected into and aspirated from the oviduct repeatedly. The flushing solution was centrifuged to separate the fluid from the cellular debris. Proteins were identified by nano-LC-MS/MS. Two different stages of the estrous cycle (Day 1 and Day 3) were analyzed in samples from 30 heifers. Both methods were applied successfully and in total, more than 3000 proteins were identified, so far representing the most comprehensive OF proteome published. This new minimal invasive approach to access the bovine oviductal fluid proteome facilitates future innovative experimental designs to study the role of the oviductal micro environment during early embryo development.


Assuntos
Líquidos Corporais/química , Bovinos , Endoscopia/veterinária , Tubas Uterinas/fisiologia , Proteoma/química , Animais , Cromatografia Líquida , Endoscopia/métodos , Ciclo Estral/metabolismo , Feminino , Regulação da Expressão Gênica , Proteínas/química , Proteínas/metabolismo , Proteoma/metabolismo , Espectrometria de Massas em Tandem
10.
Infect Immun ; 87(6)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30962402

RESUMO

Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_27925), α1-giardin, and α11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.


Assuntos
Antígenos de Protozoários/imunologia , Giardia lamblia/imunologia , Giardíase/parasitologia , Proteoma/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Reações Cruzadas , Feminino , Giardia lamblia/química , Giardia lamblia/genética , Giardíase/imunologia , Giardíase/prevenção & controle , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteoma/química , Proteoma/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Vacinas Protozoárias/química , Vacinas Protozoárias/genética , Adulto Jovem
11.
Methods Mol Biol ; 1958: 237-261, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945222

RESUMO

Proteins with prion-like behavior are attracting an increasing interest, since accumulating evidences indicate that they play relevant roles both in health and disease. The self-assembly of these proteins into insoluble aggregates is associated with severe neuropathological processes such as amyotrophic lateral sclerosis (ALS). However, in normal conditions, they are known to accomplish a wide range of functional roles. The conformational duality of prion-like proteins is often encoded in specific protein regions, named prion-like domains (PrLDs). PrLDs are usually long and disordered regions of low complexity. We have shown that PrLDs might contain soft-amyloid cores that contribute significantly to trigger their aggregation, as well as to support their propagation. Further exploration of the role of these sequences in the conformational conversion of prion-like proteins might provide novel insights into the mechanism of action and regulation of these polypeptides, enabling the future development of therapeutic strategies. Here, we describe a set of methodologies aimed to identify and characterize these short amyloid stretches in a protein or proteome of interest, ranging from in silico detection to in vitro and in vivo evaluation and validation.


Assuntos
Biologia Molecular/métodos , Proteínas Priônicas/química , Príons/química , Sequência de Aminoácidos/genética , Amiloide/química , Amiloide/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Humanos , Proteínas Priônicas/genética , Príons/genética , Agregados Proteicos/genética , Domínios Proteicos/genética , Proteoma/química , Proteoma/genética
12.
Photochem Photobiol Sci ; 18(9): 2270-2280, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30900698

RESUMO

A benzothiophene-substituted chromenone with promising activity against Leishmania and Trypanosoma species exhibits peculiar fluorescence properties useful for identifying its complexes with target proteins in the microorganism proteomes. The emission spectra, anisotropy and time profiles of this flavonoid strongly change when moving from the free to the protein-bound forms. The same two types of emission are observed in organic solvents and their mixtures with water, with the relative band intensities depending on the solvent ability to establish hydrogen bonds with the solute. The regular emission prevails in protic solvents, while in aprotic solvents the anomalously red-shifted emission occurs from a zwitterionic tautomeric form, produced in the excited state by proton transfer within the intramolecularly H-bonded form. This interpretation finds support from an experimental and theoretical investigation of the conformational preferences of this compound in the ground and lowest excited state, with a focus on the relative twisting about the chromenone-benzothiophene interconnecting bond. An analysis of the absorption and emission spectra and of the photophysical properties of the two emitting tautomers highlights the relevance of the local microenvironment, particularly of the intra- and intermolecular hydrogen bonds in which this bioactive compound is involved, in determining both its steady-state and time-resolved fluorescence behaviour.


Assuntos
Teoria da Densidade Funcional , Flavonoides/química , Flavonoides/farmacologia , Fluorescência , Proteoma/antagonistas & inibidores , Prótons , Proteínas de Protozoários/antagonistas & inibidores , Ligações de Hidrogênio , Leishmania/efeitos dos fármacos , Estrutura Molecular , Proteoma/química , Proteínas de Protozoários/química , Trypanosoma/efeitos dos fármacos
13.
Mol Cell ; 74(2): 310-319.e7, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30852061

RESUMO

Multi-domain proteins, containing several structural units within a single polypeptide, constitute a large fraction of all proteomes. Co-translational folding is assumed to simplify the conformational search problem for large proteins, but the events leading to correctly folded, functional structures remain poorly characterized. Similarly, how the ribosome and molecular chaperones promote efficient folding remains obscure. Using optical tweezers, we have dissected early folding events of nascent elongation factor G, a multi-domain protein that requires chaperones for folding. The ribosome and the chaperone trigger factor reduce inter-domain misfolding, permitting folding of the N-terminal G-domain. Successful completion of this step is a crucial prerequisite for folding of the next domain. Unexpectedly, co-translational folding does not proceed unidirectionally; emerging unfolded polypeptide can denature an already-folded domain. Trigger factor, but not the ribosome, protects against denaturation. The chaperone thus serves a previously unappreciated function, helping multi-domain proteins overcome inherent challenges during co-translational folding.


Assuntos
Fator G para Elongação de Peptídeos/química , Biossíntese de Proteínas , Conformação Proteica , Dobramento de Proteína , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Pinças Ópticas , Fator G para Elongação de Peptídeos/genética , Peptídeos/química , Peptídeos/genética , Domínios Proteicos/genética , Proteoma/química , Proteoma/genética , Ribossomos/química , Ribossomos/genética
14.
Benef Microbes ; 10(4): 463-472, 2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-30882241

RESUMO

Specific adherence is the first requisite that a microorganism has to fulfil to become established onto a mucosal surface. It was previously shown that the OppA surface protein of Lactobacillus salivarius Lv72 bound HeLa cell cultures through interaction with glycosaminoglycans (GAGs). To determine whether this is a peculiarity of that strain or whether it can be extended to other lactobacilli, 12 strains, belonging to six species, were confronted with HeLa-cell cultures in the presence of soluble GAGs. Interference was observed to six of them, heparan sulphate and chondroitin sulphate C being more interfering than chondroitin sulphate A or chondroitin sulphate B. Furthermore, inhibition of the biosynthesis of GAGs or their elimination from the cell surface with specific enzymes also resulted in reduced adherence. Analysis of the surface proteome of Lactobacillus crispatus Lv25 and of Lactobacillus reuteri RC14 revealed single proteins that immunoreacted with antibodies raised against OppA, the main adhesin of L. salivarius Lv72. Upon MALDI-TOF-TOF analysis, they were identified as OppA-like proteins, thus indicating that these proteins participate as adhesins in attachment of diverse lactobacilli to the surface of human epithelial cells.


Assuntos
Adesinas Bacterianas/metabolismo , Células Epiteliais/metabolismo , Glicosaminoglicanos/metabolismo , Lactobacillus/metabolismo , Adesinas Bacterianas/química , Motivos de Aminoácidos , Aderência Bacteriana/efeitos dos fármacos , Glicosaminoglicanos/antagonistas & inibidores , Glicosaminoglicanos/farmacologia , Células HeLa , Humanos , Lactobacillus/genética , Proteoma/química , Proteoma/metabolismo , Rodaminas/farmacologia
15.
Nat Commun ; 10(1): 1155, 2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30858367

RESUMO

Adenosine triphosphate (ATP) plays fundamental roles in cellular biochemistry and was recently discovered to function as a biological hydrotrope. Here, we use mass spectrometry to interrogate ATP-mediated regulation of protein thermal stability and protein solubility on a proteome-wide scale. Thermal proteome profiling reveals high affinity interactions of ATP as a substrate and as an allosteric modulator that has widespread influence on protein complexes and their stability. Further, we develop a strategy for proteome-wide solubility profiling, and discover ATP-dependent solubilization of at least 25% of the insoluble proteome. ATP increases the solubility of positively charged, intrinsically disordered proteins, and their susceptibility for solubilization varies depending on their localization to different membrane-less organelles. Moreover, a few proteins, exhibit an ATP-dependent decrease in solubility, likely reflecting polymer formation. Our data provides a proteome-wide, quantitative insight into how ATP influences protein structure and solubility across the spectrum of physiologically relevant concentrations.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteoma/metabolismo , Trifosfato de Adenosina/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Células Jurkat , Espectrometria de Massas , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteoma/química , Proteômica/métodos , Solubilidade
16.
Artigo em Chinês | MEDLINE | ID: mdl-30884586

RESUMO

Objective: To investigate the changes in mass spectrometry of proteins in patients with 1-bromopropane (1-BP) poisoning after treatment and their biological functions. Methods: From May 2016 to December 2017, 3 male patients aged 31-47 years with 1-BP poisoning in Bao'an District of Shenzhen, China were enrolled in this study. The whole blood sample (2 ml) was collected before and after treatment. Label-free mass spectrometry-based proteomics was used for protein identification and quantification. The differentially expressed proteins after treatment were analyzed. Bioinformatics tools were used to analyze the functions of the identified proteins and the biological processes they were involved in. Results: Proteomic analysis showed that there were 47 proteins that were differentially expressed more than 2-fold (P<0.05) after treatment in the patients with 1-BP poisoning; of them, 27 were up-regulated and 20 were down-regulated in the serum of treated patients. The identified proteins were mainly involved in proteolysis, protein modification, immune response, complement activation, lipoprotein metabolism, signal transduction, and coagulation. Conclusion: The differentially expressed proteins after treatment can help with the diagnosis, treatment, and prognosis monitoring of 1-BP poisoning and provide potential therapeutic and prognostic markers for 1-BP poisoning treatment.


Assuntos
Proteoma/química , Adulto , China , Humanos , Hidrocarbonetos Bromados/envenenamento , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica , Resultado do Tratamento
17.
Nat Commun ; 10(1): 990, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30824702

RESUMO

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.


Assuntos
Biologia Molecular/métodos , Fenol/química , Proteínas de Ligação a RNA/isolamento & purificação , Tolueno/química , Animais , Sequência de Bases , Encéfalo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/isolamento & purificação , Proteoma/química , Proteômica/métodos , RNA/química , RNA Mensageiro , Proteínas de Ligação a RNA/química , Ribonucleoproteínas/química , Ribonucleoproteínas/isolamento & purificação , Salmonella typhimurium , Sensibilidade e Especificidade
18.
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
19.
Proc Natl Acad Sci U S A ; 116(9): 3636-3645, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30733291

RESUMO

From an abstract, informational perspective, protein domains appear analogous to words in natural languages in which the rules of word association are dictated by linguistic rules, or grammar. Such rules exist for protein domains as well, because only a small fraction of all possible domain combinations is viable in evolution. We employ a popular linguistic technique, n-gram analysis, to probe the "proteome grammar"-that is, the rules of association of domains that generate various domain architectures of proteins. Comparison of the complexity measures of "protein languages" in major branches of life shows that the relative entropy difference (information gain) between the observed domain architectures and random domain combinations is highly conserved in evolution and is close to being a universal constant, at ∼1.2 bits. Substantial deviations from this constant are observed in only two major groups of organisms: a subset of Archaea that appears to be cells simplified to the limit, and animals that display extreme complexity. We also identify the n-grams that represent signatures of the major branches of cellular life. The results of this analysis bolster the analogy between genomes and natural language and show that a "quasi-universal grammar" underlies the evolution of domain architectures in all divisions of cellular life. The nearly universal value of information gain by the domain architectures could reflect the minimum complexity of signal processing that is required to maintain a functioning cell.


Assuntos
Evolução Molecular , Domínios Proteicos/genética , Estrutura Terciária de Proteína , Proteoma/química , Archaea/química , Archaea/genética , Feminino , Humanos , Linguística , Masculino , Filogenia , Proteoma/genética
20.
Eur J Pharm Sci ; 132: 1-17, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-30797936

RESUMO

Global emergence of Tigecycline resistant Acinetobacter baumannii (TRAB) is on the horizon and poses a very serious threat to human health. There is a pressing demand for suitable therapeutics against this pathogen, particularly a vaccine to protect against TRAB infections. We present a comprehensive investigation of the complete proteome of a TRAB AB031 strain to predict promiscuous antigenic, non-allergenic, virulent B-cell derived T-cell epitopes and formulate a multi-epitope vaccine against the pathogen. We identified epitopes from three proteins: outer membrane protein assembly factor (BamA), fimbrial biogenesis outer membrane usher protein (FimD) and type IV secretion protein (Rhs) that are appropriate for vaccine design. These proteins constitute the core proteome of the pathogen, are essential, localized at the pathogen surface, non-homologous to humans, mice and to the beneficial probiotic bacteria that reside the human gut. Moreover, these proteins are ideal candidates for experimental investigation as they have favorable physicochemical properties and have strong cellular interacting networks. The predicted epitopes: FPLNDKPGD (BamA), FVHAEEAAA (FimD) and YVVAGTAAA (Rhs) have exo-membrane topology for efficient recognition of the host immune system and high affinity for the most prevalent allele in human populations, the DRB*0101. These epitopes were linked and attached to an adjuvant to enhance its antigenicity. The multi-epitope vaccine-construct was docked with the TLR4 receptor to assess its affinity for the protein and thus its presentation to the host immune system. Docking results were validated through molecular dynamics simulations and binding free energies were calculated using the molecular mechanics/generalized Born (MM-GBSA) method. In conclusion, we expect the designed chimeric vaccine is highly likely to be effective against infections caused by TRAB.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Vacinas Bacterianas/química , Biologia Computacional , Descoberta de Drogas/métodos , Epitopos/química , Acinetobacter baumannii/química , Acinetobacter baumannii/imunologia , Antibacterianos/farmacologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Farmacorresistência Bacteriana , Mapeamento de Epitopos , Epitopos/imunologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteoma/química , Tigeciclina/farmacologia , Receptor 4 Toll-Like/química , Vacinas de Subunidades/química , Vacinas de Subunidades/imunologia
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