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1.
Ecotoxicol Environ Saf ; 220: 112334, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34020284

RESUMO

Artificial light at night (ALAN) is a widespread environmental pollutant and stressor. Many nocturnal insects have been shown to experience ALAN stress. However, few studies have been conducted to uncover the mechanism by which nocturnal insects respond to ALAN stress. Previous studies suggest that lysine succinylation (Ksuc) is a potential mechanism that coordinates energy metabolism and antioxidant activity under stressful conditions. Mythimna separata (Walker) (M. separata) is a nocturnal insect that has been stressed by ALAN. In this study, we quantified the relative proteomic Ksuc levels in ALAN-stressed M. separata. Of the 466 identified Ksuc-modified proteins, 103 were hypersuccinylated/desuccinylated in ALAN-stressed moths. The hypersuccinylated/desuccinylated proteins were shown to be involved in various biological processes. In particular, they were enriched in metabolic processes, reactive oxygen species (ROS) homeostasis and the neuromuscular system. Furthermore, we demonstrated that Ksuc might affect moth locomotion by intervening with and coordinating these systems under ALAN stress. These findings suggest that Ksuc plays a vital role in the moth response to ALAN stress and moth locomotion behavior and provide a new perspective on the impact of ALAN on nocturnal insect populations and species communities.


Assuntos
Proteínas de Insetos/química , Luz , Iluminação , Lisina/química , Mariposas/fisiologia , Fototaxia , Proteoma/química , Animais , Antioxidantes/metabolismo , Metabolismo Energético , Estresse Fisiológico
2.
Nature ; 594(7862): 246-252, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33845483

RESUMO

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-ß pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.


Assuntos
COVID-19/metabolismo , Interações Hospedeiro-Patógeno , Proteoma/metabolismo , Proteômica , Vírus da SARS/patogenicidade , SARS-CoV-2/patogenicidade , Síndrome Respiratória Aguda Grave/metabolismo , Animais , Antivirais/farmacologia , Autofagia/efeitos dos fármacos , COVID-19/imunologia , COVID-19/virologia , Linhagem Celular , Conjuntos de Dados como Assunto , Avaliação Pré-Clínica de Medicamentos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Inibidores de Metaloproteinases de Matriz/farmacologia , Fosforilação , Mapas de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteoma/química , Vírus da SARS/imunologia , SARS-CoV-2/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinação , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Viroporinas/metabolismo
3.
Molecules ; 26(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652646

RESUMO

Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3-5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children.


Assuntos
Vesículas Extracelulares/genética , Proteoma/genética , Proteômica , Urina/química , Aminopeptidases/urina , Proteínas Semelhantes a Angiopoietina/urina , Pré-Escolar , Dipeptidil Peptidases e Tripeptidil Peptidases/urina , Impedância Elétrica , Proteínas do Olho/urina , Feminino , Humanos , Masculino , Fatores de Crescimento Neural/urina , Proteoma/química , Receptores de Superfície Celular/genética , Testes de Função Respiratória , Serina Proteases/urina , Serpinas/urina , Antígenos Thy-1/urina
4.
Food Chem ; 352: 129436, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33691214

RESUMO

This study aimed to investigate changes in macronutrients, total bacterial count, and serum proteome of human milk (HM) under different frozen storage (-18°C and -60°C, 60 d and 180 d) by using IBT Labeling proteomics techniques and ELISA kit. The results indicated that total protein concentrations and total aerobic bacterial counts were significantly decreased at -18°C, while no difference at -60°C. A total of 1617 proteins were identified and quantified, and 173 proteins were significantly different. The -18°C storage had much higher influence on HM serum protein profiles than that of -60°C. Increased milk fat globule membrane (MFGM) proteins at -18°C are highly related to the damage of MFGM and transfer of MFGM proteins. The reduction of bioactive proteins is probably related to the ice-induced denaturation. In conclusion, fast cooling and ultra-low constant temperature are more suitable for the cryopreservation of human milk.


Assuntos
Armazenamento de Alimentos , Congelamento , Proteínas do Leite/análise , Leite Humano/química , Proteoma/química , Soro/química , Animais , Humanos
5.
J Mol Biol ; 433(10): 166944, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33741411

RESUMO

Genome-wide protein-protein interaction (PPI) determination remains a significant unsolved problem in structural biology. The difficulty is twofold since high-throughput experiments (HTEs) have often a relatively high false-positive rate in assigning PPIs, and PPI quaternary structures are more difficult to solve than tertiary structures using traditional structural biology techniques. We proposed a uniform pipeline, Threpp, to address both problems. Starting from a pair of monomer sequences, Threpp first threads both sequences through a complex structure library, where the alignment score is combined with HTE data using a naïve Bayesian classifier model to predict the likelihood of two chains to interact with each other. Next, quaternary complex structures of the identified PPIs are constructed by reassembling monomeric alignments with dimeric threading frameworks through interface-specific structural alignments. The pipeline was applied to the Escherichia coli genome and created 35,125 confident PPIs which is 4.5-fold higher than HTE alone. Graphic analyses of the PPI networks show a scale-free cluster size distribution, consistent with previous studies, which was found critical to the robustness of genome evolution and the centrality of functionally important proteins that are essential to E. coli survival. Furthermore, complex structure models were constructed for all predicted E. coli PPIs based on the quaternary threading alignments, where 6771 of them were found to have a high confidence score that corresponds to the correct fold of the complexes with a TM-score >0.5, and 39 showed a close consistency with the later released experimental structures with an average TM-score = 0.73. These results demonstrated the significant usefulness of threading-based homologous modeling in both genome-wide PPI network detection and complex structural construction.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Choque Térmico HSP70/genética , Fosfotransferases/genética , Proteoma/genética , Fatores de Transcrição/genética , Teorema de Bayes , Análise por Conglomerados , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Fosfotransferases/química , Fosfotransferases/metabolismo , Dobramento de Proteína , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/genética , Estrutura Quaternária de Proteína , Proteoma/química , Proteoma/metabolismo , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
6.
Acc Chem Res ; 54(7): 1801-1813, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33733731

RESUMO

One of the biggest bottlenecks in modern drug discovery efforts is in tackling the undruggable proteome. Currently, over 85% of the proteome is still considered undruggable because most proteins lack well-defined binding pockets that can be functionally targeted with small molecules. Tackling the undruggable proteome necessitates innovative approaches for ligand discovery against undruggable proteins as well as the development of new therapeutic modalities to functionally manipulate proteins of interest. Chemoproteomic platforms, in particular activity-based protein profiling (ABPP), have arisen to tackle the undruggable proteome by using reactivity-based chemical probes and advanced quantitative mass spectrometry-based proteomic approaches to enable the discovery of "ligandable hotspots" or proteome-wide sites that can be targeted with small-molecule ligands. These sites can subsequently be pharmacologically targeted with covalent ligands to rapidly discover functional or nonfunctional binders against therapeutic proteins of interest. Chemoproteomic approaches have also revealed unique insights into ligandability such as the discovery of unique allosteric sites or intrinsically disordered regions of proteins that can be pharmacologically and selectively targeted for biological modulation and therapeutic benefit. Chemoproteomic platforms have also expanded the scope of emerging therapeutic modalities for targeted protein degradation and proteolysis-targeting chimeras (PROTACs) through the discovery of several new covalent E3 ligase recruiters. Looking into the future, chemoproteomic approaches will unquestionably have a major impact in further expansion of existing efforts toward proteome-wide ligandability mapping, targeted ligand discovery efforts against high-value undruggable therapeutic targets, further expansion of the scope of targeted protein degradation platforms, the discovery of new molecular glue scaffolds that enable unique modulation of protein function, and perhaps most excitingly the development of next-generation small-molecule induced-proximity-based therapeutic modalities that go beyond degradation. Exciting days lie ahead in this field as chemical biology becomes an increasingly major driver in drug discovery, and chemoproteomic approaches are sure to be a mainstay in developing next-generation therapeutics.


Assuntos
Proteoma/química , Proteômica , Descoberta de Drogas , Humanos , Ligantes , Proteólise
7.
Nat Commun ; 12(1): 891, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563959

RESUMO

Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.


Assuntos
Metilistidinas/metabolismo , Metiltransferases/metabolismo , Proteoma/metabolismo , Motivos de Aminoácidos , Animais , Células Cultivadas , Histidina/metabolismo , Humanos , Mamíferos/classificação , Mamíferos/genética , Mamíferos/metabolismo , Metilação , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mutação , Processamento de Proteína Pós-Traducional , Proteoma/química , Especificidade por Substrato , Zinco/metabolismo
8.
Exp Parasitol ; 223: 108082, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33581108

RESUMO

Leishmaniasis is a complex vector-borne disease mediated by Leishmania parasite and a strong and long-lasting CD4+ Th1 and CD8+-T cell immunity is required to control the infection. Thus far multivalent subunit vaccines have met this requirement more promisingly. However several full protein sequences cannot be easily arranged in one construct. Instead, new emerging immune-informatics based epitope formulations surpass this restriction. Herein, we aimed to examine the protective potential of a dendritic cell based vaccine presenting epitopes to CD8+ and CD4+-T cells in combination with DNA vaccine encoding the same epitopes against murine cutaneous leishmaniasis. Immature DCs were loaded with epitopes (selected from parasite proteome) in vitro with or without CpG oligonucleotides and were used to immunize BALB/c mice. Peptide coding DNA was used to boost the system and immunological responses were evaluated after Leishmania (L.) major infectious challenge. The pre-challenge response to included epitopes was Th1 polarized which potentially lowered the infection at early time points post-challenge but not at later weeks. Collectively, DC prime-DNA boost was found to be a promising approach for Th1 polarization however the constituent epitopes undoubtedly make a significant contribution in the protection outcome of the vaccine.


Assuntos
Células da Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Epitopos/química , Epitopos/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Proteoma/química , Vacinas de DNA
9.
ACS Chem Biol ; 16(2): 389-396, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33524253

RESUMO

Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic post-translational modification regulated by various writers, readers, and erasers. It participates in a variety of biological events and is involved in many human diseases. Currently, tools and technologies have yet to be developed for unambiguously defining readers and erasers of individual PARylated proteins or cognate PARylated proteins for known readers and erasers. Here, we report the generation of a bifunctional nicotinamide adenine dinucleotide (NAD+) characterized by diazirine-modified adenine and clickable ribose. By serving as an excellent substrate for poly-ADP-ribose polymerase 1 (PARP1)-catalyzed PARylation, the generated bifunctional NAD+ enables photo-cross-linking and enrichment of PARylation-dependent interacting proteins for proteomic identification. This bifunctional NAD+ provides an important tool for mapping cellular interaction networks centered on protein PARylation, which are essential for elucidating the roles of PARylation-based signals or activities in physiological and pathophysiological processes.


Assuntos
Reagentes para Ligações Cruzadas/metabolismo , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteoma/metabolismo , Azidas/síntese química , Azidas/metabolismo , Azidas/efeitos da radiação , Química Click , Reagentes para Ligações Cruzadas/síntese química , Reagentes para Ligações Cruzadas/efeitos da radiação , Diazometano/análogos & derivados , Diazometano/metabolismo , Diazometano/efeitos da radiação , Células HEK293 , Humanos , NAD/síntese química , NAD/efeitos da radiação , Poli ADP Ribosilação , Processamento de Proteína Pós-Traducional , Proteoma/química , Proteômica , Raios Ultravioleta
10.
Artigo em Inglês | MEDLINE | ID: mdl-33412504

RESUMO

In Nanomedicine, carbon-based nanomaterials, like Carbon Nanotubes (CNT), are considered potential candidates as drug delivery systems. In vivo adsorption of physiological proteins onto carbon nanotubes, through noncovalent interactions, forms a protein corona or bio corona, able to influence biological properties and biocompatibility of CNT. This study aimed to explore the composition of protein corona formed onto PEGylated Multi-Walled Carbon Nanotubes (MWCNT-PEG5k), after their incubation in human plasma. Plasma proteins were sequentially eluted in different conditions by using both native and denaturant buffers, useful to characterize soft and hard corona. Proteomic methods and mass spectrometry analysis have identified proteins in soft corona, involved in the regulation of immune response and in the CNT transport, and biomolecules in hard corona with a role in the maintenance of host homeostasis. These promising results have demonstrated the potential of PEGylated Multi-Walled Carbon Nanotubes as future candidates for drug delivery.


Assuntos
Nanotubos de Carbono , Coroa de Proteína , Proteômica/métodos , Adsorção , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Cromatografia Líquida , Sistemas de Liberação de Medicamentos , Humanos , Nanotubos de Carbono/análise , Nanotubos de Carbono/química , Polietilenoglicóis/química , Coroa de Proteína/análise , Coroa de Proteína/química , Proteoma/análise , Proteoma/química , Proteoma/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
11.
Artigo em Inglês | MEDLINE | ID: mdl-33486215

RESUMO

Nascent proteome presents dynamic changes in response to a certain stimulus. Thus, monitoring nascent proteome is critical to uncovering the involved biological mechanism. But the low-abundance of nascent proteome against an overwhelming pre-existing proteome limits its identification and quantification. Herein, we present a novel strategy to enrich nascent proteome from whole cell lysate for further analysis by mass spectrometry. We employed a terminal alkyne and disulfide functionalized agarose resin to capture nascent proteome which had been labeled by L-azidohomoalanine. Results from the western blot, silver staining and pulse metabolic labeling suggested that the nascent proteome could be enriched efficiently. Applied to Hela cells, the method identified about 700 nascent proteins with good correlation with previous reports. The above indicates that our strategy can be used to reveal the proteome dynamics of biological processes.


Assuntos
Alanina/análogos & derivados , Proteínas/análise , Sefarose/química , Alanina/química , Alcinos/química , Cromatografia Líquida , Química Click , Dissulfetos/química , Células HeLa , Humanos , Proteínas/química , Proteínas/metabolismo , Proteoma/análise , Proteoma/química , Proteoma/metabolismo , Espectrometria de Massas em Tandem
12.
Nucleic Acids Res ; 49(D1): D309-D318, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-32976589

RESUMO

Alternative splicing plays a major role in regulating the functional repertoire of the proteome. However, isoform-specific effects to protein-protein interactions (PPIs) are usually overlooked, making it impossible to judge the functional role of individual exons on a systems biology level. We overcome this barrier by integrating protein-protein interactions, domain-domain interactions and residue-level interactions information to lift exon expression analysis to a network level. Our user-friendly database DIGGER is available at https://exbio.wzw.tum.de/digger and allows users to seamlessly switch between isoform and exon-centric views of the interactome and to extract sub-networks of relevant isoforms, making it an essential resource for studying mechanistic consequences of alternative splicing.


Assuntos
Processamento Alternativo , Bases de Dados de Proteínas , Éxons , Mapeamento de Interação de Proteínas/métodos , Proteoma/química , RNA Mensageiro/genética , Sítios de Ligação , Biologia Computacional/métodos , Humanos , Internet , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Proteoma/genética , Proteoma/metabolismo , RNA Mensageiro/metabolismo , Software , Termodinâmica
13.
Nucleic Acids Res ; 49(D1): D298-D308, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33119734

RESUMO

We present DescribePROT, the database of predicted amino acid-level descriptors of structure and function of proteins. DescribePROT delivers a comprehensive collection of 13 complementary descriptors predicted using 10 popular and accurate algorithms for 83 complete proteomes that cover key model organisms. The current version includes 7.8 billion predictions for close to 600 million amino acids in 1.4 million proteins. The descriptors encompass sequence conservation, position specific scoring matrix, secondary structure, solvent accessibility, intrinsic disorder, disordered linkers, signal peptides, MoRFs and interactions with proteins, DNA and RNAs. Users can search DescribePROT by the amino acid sequence and the UniProt accession number and entry name. The pre-computed results are made available instantaneously. The predictions can be accesses via an interactive graphical interface that allows simultaneous analysis of multiple descriptors and can be also downloaded in structured formats at the protein, proteome and whole database scale. The putative annotations included by DescriPROT are useful for a broad range of studies, including: investigations of protein function, applied projects focusing on therapeutics and diseases, and in the development of predictors for other protein sequence descriptors. Future releases will expand the coverage of DescribePROT. DescribePROT can be accessed at http://biomine.cs.vcu.edu/servers/DESCRIBEPROT/.


Assuntos
Aminoácidos/química , Bases de Dados de Proteínas , Genoma , Proteínas/genética , Proteoma/genética , Software , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Bactérias/metabolismo , Sítios de Ligação , Sequência Conservada , Fungos/genética , Fungos/metabolismo , Humanos , Internet , Plantas/genética , Plantas/metabolismo , Células Procarióticas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/classificação , Proteínas/metabolismo , Proteoma/química , Proteoma/metabolismo , Análise de Sequência de Proteína , Vírus/genética , Vírus/metabolismo
14.
Protein Sci ; 30(1): 218-233, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33146890

RESUMO

For a complete understanding of a system's processes and each protein's role in health and disease, it is essential to study protein expression with a spatial resolution, as the exact location of proteins at tissue, cellular, or subcellular levels is tightly linked to protein function. The Human Protein Atlas (HPA) project is a large-scale initiative aiming at mapping the entire human proteome using antibody-based proteomics and integration of various other omics technologies. The publicly available knowledge resource www.proteinatlas.org is one of the world's most visited biological databases and has been extensively updated during the last few years. The current version is divided into six main sections, each focusing on particular aspects of the human proteome: (a) the Tissue Atlas showing the distribution of proteins across all major tissues and organs in the human body; (b) the Cell Atlas showing the subcellular localization of proteins in single cells; (c) the Pathology Atlas showing the impact of protein levels on survival of patients with cancer; (d) the Blood Atlas showing the expression profiles of blood cells and actively secreted proteins; (e) the Brain Atlas showing the distribution of proteins in human, mouse, and pig brain; and (f) the Metabolic Atlas showing the involvement of proteins in human metabolism. The HPA constitutes an important resource for further understanding of human biology, and the publicly available datasets hold much promise for integration with other emerging efforts focusing on single cell analyses, both at transcriptomic and proteomic level.


Assuntos
Encefalopatias/metabolismo , Encéfalo/metabolismo , Bases de Dados de Proteínas , Proteínas do Tecido Nervoso , Proteoma , Animais , Humanos , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteoma/química , Proteoma/metabolismo , Suínos
15.
Artigo em Inglês | MEDLINE | ID: mdl-33279813

RESUMO

Shotgun proteomics is a high-throughput technology which has been developed with the aim of investigating the maximum number of proteins in cells in a given experiment. However, protein discovery and data generation vary in depth and coverage when different technical strategies are selected. In this study, three different sample preparation approaches, and peptide or protein fractionation methods, were applied to identify and quantify proteins from log-phase yeast lysate: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), filter-aided sample preparation coupled with gas phase fractionation (FASP-GPF), and FASP - high pH reversed phase fractionation (HpH). Fractions were initially analyzed and compared using nanoflow liquid chromatography - tandem mass spectrometry (nanoLC-MS/MS) employing data dependent acquisition on a linear ion trap instrument. The number of fractions and analytical replicates was adjusted so that each experiment used a similar amount of mass spectrometric instrument time. A second set of experiments was performed, comparing FASP-GPF, SDS-PAGE and FASP-HpH using a Q Exactive Orbitrap mass spectrometer. Compared with results from the linear ion trap mass spectrometer, the use of a Q Exactive Orbitrap mass spectrometer enabled a substantial increase in protein identifications, and an even greater increase in peptide identifications. This shows that the main advantage of using the higher resolution instrument is in increased proteome coverage. A total of 1035, 1357 and 2134 proteins were separately identified by FASP-GPF, SDS-PAGE and FASP-HpH. Combining results from the Orbitrap experiments, there were a total of 2269 proteins found, with 94% of them identified using the FASP-HpH method. Therefore, the FASP-HpH method is the optimal choice among these approaches, when applied to this type of sample.


Assuntos
Cromatografia de Fase Reversa/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Concentração de Íons de Hidrogênio , Peptídeos/análise , Peptídeos/química , Proteoma/análise , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Espectrometria de Massas em Tandem/métodos
16.
Nucleic Acids Res ; 49(D1): D1548-D1554, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33174598

RESUMO

Lectins are non-covalent glycan-binding proteins mediating cellular interactions but their annotation in newly sequenced organisms is lacking. The limited size of functional domains and the low level of sequence similarity challenge usual bioinformatics tools. The identification of lectin domains in proteomes requires the manual curation of sequence alignments based on structural folds. A new lectin classification is proposed. It is built on three levels: (i) 35 lectin domain folds, (ii) 109 classes of lectins sharing at least 20% sequence similarity and (iii) 350 families of lectins sharing at least 70% sequence similarity. This information is compiled in the UniLectin platform that includes the previously described UniLectin3D database of curated lectin 3D structures. Since its first release, UniLectin3D has been updated with 485 additional 3D structures. The database is now complemented by two additional modules: PropLec containing predicted ß-propeller lectins and LectomeXplore including predicted lectins from sequences of the NBCI-nr and UniProt for every curated lectin class. UniLectin is accessible at https://www.unilectin.eu/.


Assuntos
Bases de Dados de Proteínas , Genoma , Lectinas/química , Proteoma/química , Receptores de Superfície Celular/química , Sequência de Aminoácidos , Animais , Antozoários/genética , Antozoários/metabolismo , Biologia Computacional/métodos , Humanos , Internet , Lectinas/classificação , Lectinas/genética , Lectinas/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteoma/classificação , Proteoma/genética , Proteoma/metabolismo , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Software , Terminologia como Assunto
17.
Toxicon ; 189: 91-104, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33181162

RESUMO

Several research groups have studied the components produced by the venom gland of the scorpion Tityus serrulatus, which has one of the most lethal venoms in the world. Various methodologies have been employed to clarify the complex mechanisms of action of these components, especially neurotoxins and enzymes. Transcriptomes and proteomes have provided important information for pharmacological, biochemical, and immunological research. Next-generation sequencing (NGS) has allowed the description of new transcripts and completion of partial sequence descriptions for peptides, especially those with low expression levels. In the present work, after NGS sequencing, we searched for new putative venom components. We present a total of nine new transcripts with neurotoxic potential (Ts33-41) and describe the sequences of one hyaluronidase (TsHyal_4); three enzymes involved in amidation (peptidyl-glycine alpha-amidating monooxygenase A, peptidyl-alpha-hydroxyglycine alpha-amidating lyase, and peptidylglycine alpha-hydroxylating monooxygenase), which increases the lethal potential of neurotoxins; and also the enzyme Ts_Chitinase1, which may be involved in the venom's digestive action. In addition, we determined the level of transcription of five groups: toxins, metalloproteases, hyaluronidases, chitinases and amidation enzymes, including new components found in this study. Toxins are the predominant group with an expression level of 91.945%, followed by metalloproteases with only 7.790% and other groups representing 0.265%.


Assuntos
Proteoma/química , Venenos de Escorpião/química , Escorpiões , Amidina-Liases , Sequência de Aminoácidos , Animais , Biologia Computacional , Metaloproteases , Oxigenases de Função Mista , Complexos Multienzimáticos , Transcriptoma
18.
Methods Mol Biol ; 2237: 45-53, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237407

RESUMO

Because of narrow availability of antibody pairs and potential cross-reactivity between antibodies, the development of sandwich-based antibody arrays which need a pair of antibodies for each target has been restricted to higher density resulting in limited proteomic breadth of detection. Label-based array is one way to overcome this obstacle by directly labeling all targets in samples with fluorescent dyes such as Cy3 and Cy5. The labeled samples are then applied on the antibody array chip composed of capture antibodies. In this chapter, we will introduce this technology including array production and sample detection assay.


Assuntos
Imunofluorescência/métodos , Análise Serial de Proteínas/métodos , Proteômica/métodos , Animais , Biotinilação/métodos , Corantes Fluorescentes/química , Humanos , Imunoensaio/métodos , Proteoma/química , Proteoma/imunologia
19.
Methods Mol Biol ; 2130: 185-193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33284445

RESUMO

Recent advances in mass spectrometry (MS)-based quantitative proteomics now allow the identification and quantification of deep proteomes and post-translational modifications (PTMs) in relatively short times. Therefore, in the last few years, this technology has proven successful in the circadian field to characterize temporal oscillations of the proteome and more recently PTMs in cellular systems and in tissues. In this chapter, we describe a robust and simple protocol, based on the EasyPhos workflow, to enable preparation of large number of proteomes and phosphoproteomes from mouse tissues for MS-based quantitative analysis. We additionally discuss computational methods to analyze proteome and phosphoproteome time series to determine circadian oscillations.


Assuntos
Ritmo Circadiano , Espectrometria de Massas/métodos , Fosfoproteínas/química , Proteômica/métodos , Animais , Camundongos , Fosfoproteínas/metabolismo , Proteoma/química , Proteoma/metabolismo
20.
J Agric Food Chem ; 69(1): 568-583, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33371680

RESUMO

The hull-less barley (Qingke) is widely planted as a staple food crop in the Tibetan area, China, and the grains contains high content of ß-glucan (BG). The mechanisms of BG synthesis and accumulation in qingke has not been studied at the protein level. This study characterized the proteins associated with BG synthesis and accumulation during qingke seed development. The proteome profiles of qingke seeds taken at 20, 30, and 40 days after flowering were compared using the TMT-based quantitative proteomics. A total of 4283 proteins were identified, with 759 being differentially expressed (DEPs) throughout seed development. Comparisons of protein expression pattern, functions, and pathway enrichment tests highlight cell wall modification, carbon and energy metabolism, polysaccharide metabolism, post-transcriptional modifications, and vesicular transport as critical biological processes related to qingke BG accumulation. Furthermore, induction of starch synthase, starch branching enzyme, pectin acetyl esterases, beta-glucosidases, beta-amylases, 1,4-beta-xylan, xyloglucan, α-amylase inhibitors, and glycosyltransferases underpinned BG synthesis. The results also indicated that the proteins involved in glycolytic, gluconeogenesis, and glyoxylate bypass pathways provided energy and reducing power for BG storage. Parallel reaction monitoring (PRM) and quantitative real-time PCR (qPCR) analyses confirmed the expression profile of the proteins obtained by TMT-based proteomics. The current results provided an insight into the mechanisms of BG synthesis and accumulation during qingke seed development.


Assuntos
Hordeum/genética , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , beta-Glucanas/metabolismo , China , Regulação da Expressão Gênica de Plantas , Hordeum/química , Hordeum/crescimento & desenvolvimento , Hordeum/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Proteômica , Sementes/química , Sementes/genética , Sementes/metabolismo , beta-Glucanas/química
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