RESUMO
Deregulation of mitochondrial functions in hepatocytes contributes to many liver diseases, such as nonalcoholic fatty liver disease (NAFLD). Lately, it was referred to as MAFLD (metabolism-associated fatty liver disease). Hesperetin (Hst), a bioactive flavonoid constituent of citrus fruit, has been proven to attenuate NAFLD. However, a potential connection between its preventive activities and the modulation of mitochondrial functions remains unclear. Here, our results showed that Hst alleviates palmitic acid (PA)-triggered NLRP3 inflammasome activation and cell death by inhibition of mitochondrial impairment in HepG2 cells. Hst reinstates fatty acid oxidation (FAO) rates measured by seahorse extracellular flux analyzer and intracellular acetyl-CoA levels as well as intracellular tricarboxylic acid cycle metabolites levels including NADH and FADH2 reduced by PA exposure. In addition, Hst protects HepG2 cells against PA-induced abnormal energetic profile, ATP generation reduction, overproduction of mitochondrial reactive oxygen species, and collapsed mitochondrial membrane potential. Furthermore, Hst improves the protein expression involved in PINK1/Parkin-mediated mitophagy. Our results demonstrate that it restores PA-impaired mitochondrial function and sustains cellular homeostasis due to the elevation of PINK1/Parkin-mediated mitophagy and the subsequent disposal of dysfunctional mitochondria. These results provide therapeutic potential for Hst utilization as an effective intervention against fatty liver disease.
Assuntos
Hesperidina , Mitocôndrias , Mitofagia , Ácido Palmítico , Proteínas Quinases , Ubiquitina-Proteína Ligases , Humanos , Células Hep G2 , Ácido Palmítico/farmacologia , Hesperidina/farmacologia , Mitofagia/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Substâncias Protetoras/farmacologiaRESUMO
Sulfur is an essential nutrient for all plants, but also crucial for the nitrogen fixing symbiosis between legumes and rhizobia. Sulfur limitation can hamper nodule development and functioning. Until now, it remained unclear whether sulfate uptake into nodules is local or mainly systemic via the roots, and if long-distance transport from shoots to roots and into nodules occurs. Therefore, this work investigates the systemic regulation of sulfur transportation in the model legume Lotus japonicus by applying stable isotope labeling to a split-root system. Metabolite and protein extraction together with mass spectrometry analyses were conducted to determine the plants molecular phenotype and relative isotope protein abundances. Data show that treatments of varying sulfate concentrations including the absence of sulfate on one side of a nodulated root was not affecting nodule development as long as the other side of the root system was provided with sufficient sulfate. Concentrations of shoot metabolites did not indicate a significant stress response caused by a lack of sulfur. Further, we did not observe any quantitative changes in proteins involved in biological nitrogen fixation in response to the different sulfate treatments. Relative isotope abundance of 34S confirmed a long-distance transport of sulfur from one side of the roots to the other side and into the nodules. Altogether, these results provide evidence for a systemic long-distance transport of sulfur via the upper part of the plant to the nodules suggesting a demand driven sulfur distribution for the maintenance of symbiotic N-fixation.
Assuntos
Lotus , Proteínas de Plantas , Nódulos Radiculares de Plantas , Enxofre , Simbiose , Nódulos Radiculares de Plantas/metabolismo , Enxofre/metabolismo , Proteínas de Plantas/metabolismo , Lotus/metabolismo , Transporte Biológico , Fixação de Nitrogênio , Sulfatos/metabolismo , Raízes de Plantas/metabolismoRESUMO
Stress Knowledge Map (SKM; https://skm.nib.si) is a publicly available resource containing two complementary knowledge graphs that describe the current knowledge of biochemical, signaling, and regulatory molecular interactions in plants: a highly curated model of plant stress signaling (PSS; 543 reactions) and a large comprehensive knowledge network (488 390 interactions). Both were constructed by domain experts through systematic curation of diverse literature and database resources. SKM provides a single entry point for investigations of plant stress response and related growth trade-offs, as well as interactive explorations of current knowledge. PSS is also formulated as a qualitative and quantitative model for systems biology and thus represents a starting point for a plant digital twin. Here, we describe the features of SKM and show, through two case studies, how it can be used for complex analyses, including systematic hypothesis generation and design of validation experiments, or to gain new insights into experimental observations in plant biology.
Assuntos
Plantas , Estresse Fisiológico , Biologia de Sistemas , Plantas/genética , Plantas/metabolismo , Fenômenos Fisiológicos Vegetais/genética , Transdução de Sinais/genética , Bases de Dados FactuaisRESUMO
Phosphoglycerate dehydrogenase (PHGDH) has emerged as a crucial factor in macromolecule synthesis, neutralizing oxidative stress, and regulating methylation reactions in cancer cells, lymphocytes, and endothelial cells. However, the role of PHGDH in tumor-associated macrophages (TAMs) is poorly understood. Here, we found that the T helper 2 (Th2) cytokine interleukin-4 and tumor-conditioned media upregulate the expression of PHGDH in macrophages and promote immunosuppressive M2 macrophage activation and proliferation. Loss of PHGDH disrupts cellular metabolism and mitochondrial respiration, which are essential for immunosuppressive macrophages. Mechanistically, PHGDH-mediated serine biosynthesis promotes α-ketoglutarate production, which activates mTORC1 signaling and contributes to the maintenance of an M2-like macrophage phenotype in the tumor microenvironment. Genetic ablation of PHGDH in macrophages from tumor-bearing mice results in attenuated tumor growth, reduced TAM infiltration, a phenotypic shift of M2-like TAMs toward an M1-like phenotype, downregulated PD-L1 expression and enhanced antitumor T-cell immunity. Our study provides a strong basis for further exploration of PHGDH as a potential target to counteract TAM-mediated immunosuppression and hinder tumor progression.
Assuntos
Ácidos Cetoglutáricos , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosfoglicerato Desidrogenase , Transdução de Sinais , Microambiente Tumoral , Macrófagos Associados a Tumor , Animais , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfoglicerato Desidrogenase/metabolismo , Camundongos , Ácidos Cetoglutáricos/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Fenótipo , Linhagem Celular Tumoral , Ativação de MacrófagosRESUMO
Dopamine (DA) is critically involved in different functions of the central nervous system (CNS) including control of voluntary movement, affect, reward, sleep, and cognition. One of the key components of DA neurotransmission is DA reuptake by the DA transporter (DAT), ensuring rapid clearance of DA from the synaptic cleft. Thus, lack of DAT leads to persistent high extracellular DA levels. While there is strong evidence for a role of striatal dopaminergic activity in learning and memory processes, little is known about the contribution of DAT deficiency to conditional learning impairments and underlying molecular processes. DAT-knockout (DAT-KO) rats were tested in a set of behavioral experiments evaluating conditional associative learning, which requires unaltered striatal function. In parallel, a large-scale proteomic analysis of the striatum was performed to identify molecular factors probably underlying behavioral patterns. DAT-KO rats were incapable to acquire a new operant skill in Pavlovian/instrumental autoshaping, although the conditional stimulus-unconditional stimulus (CS-US) association seems to be unaffected. These findings suggest that DAT directly or indirectly contributes to the reduction of transference of incentive salience from the reward to the CS. We propose that specific impairment of conditional learning might be caused by molecular adaptations to the hyperdopaminergic state, presumably by dopamine receptor 1 (DRD1) hypofunction, as proposed by proteomic analysis. Whether DRD1 downregulation can cause cognitive deficits in the hyperdopaminergic state is the subject of discussion, and further studies are needed to answer this question. This study may be useful for the interpretation of previous and the design of future studies in the dopamine field.
RESUMO
MS-based proteomics has become the most utilized tool to characterize histone PTMs. Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (<6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization. However, the propionyl group is not sufficiently hydrophobic to fully retain the shortest histone peptides in RP LC, and such procedure also hampers the discovery of natural propionylation events. In this work we tested 12 commercially available anhydrides, selected based on their safety and hydrophobicity. Performance was evaluated in terms of yield of the reaction, MS/MS fragmentation efficiency, and drift in retention time using the following samples: (i) a synthetic unmodified histone H3 tail, (ii) synthetic modified histone peptides, and (iii) a histone extract from cell lysate. Results highlighted that seven of the selected anhydrides increased peptide retention time as compared to propionic, and several anhydrides such as benzoic and valeric led to high MS/MS spectra quality. However, propionic anhydride derivatization still resulted, in our opinion, as the best protocol to achieve high MS sensitivity and even ionization efficiency among the analyzed peptides.
Assuntos
Anidridos/química , Histonas/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Peptídeos/análiseRESUMO
To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua™ technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono- (me1), di- (me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the uncorrected data. The peptide library and detection efficiencies presented here may serve as a resource to facilitate studies in the epigenetics and proteomics fields.
Assuntos
Histonas/metabolismo , Biblioteca de Peptídeos , Aminoácidos/metabolismo , Animais , Células Cultivadas , Histonas/química , Marcação por Isótopo , Espectrometria de Massas , Metilação , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional , Células-Tronco , TrofoblastosRESUMO
Protein methylation is a post-translational modification (PTM) which modulates cellular and biological processes including transcription, RNA processing, protein interactions and protein dynamics. Methylation, catalyzed by highly specific methyltransferase enzymes, occurs on several amino acids including arginine, lysine, histidine and dicarboxylic amino acids like glutamate. Mass spectrometry (MS)-based techniques continue to be the methods of choice for the study of protein PTMs. These approaches are powerful and sensitive tools that have been used to identify, quantify and characterize protein methylation. In addition, metabolic labeling strategies can be coupled to MS detection in order to measure dynamic and differential in vivo protein methylation rates. In this review, different applications of mass spectrometry technologies and methods to study protein methylation are discussed.
Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Aminoácidos/análise , Aminoácidos/metabolismo , Animais , Histonas/química , Histonas/metabolismo , Humanos , Espectrometria de Massas/instrumentação , Metilação , Proteínas/metabolismoRESUMO
The discovery of biomarkers for neurodegenerative diseases will have a major impact on the efficiency of therapeutic clinical trials and may be important for understanding basic pathogenic mechanisms. We have approached the discovery of protein biomarkers for amyotrophic lateral sclerosis (ALS) by profiling affected tissues in a relevant animal model and then validating the findings in human tissues. Ventral roots from SOD1(G93A) "ALS" mice were analyzed by label-free quantitative mass spectrometry, and the resulting data were compared with data for matched samples from nontransgenic littermates and transgenic mice carrying wild-type human SOD1 (SOD1(WT)). Of 1299 proteins, statistical inference of the data in the three groups identified 14 proteins that were dramatically altered in the ALS mice compared with the two control groups. The protein galectin-3 emerged as a lead biomarker candidate on the basis of its differential expression as assessed by immunoblot and immunocytochemistry in SOD1(G93A) mice as compared to controls and because it is a secreted protein that could potentially be measured in human biofluids. Spinal cord tissue from ALS patients also exhibited increased levels of galectin-3 when compared to controls. Further measurement of galectin-3 in cerebrospinal fluid samples showed that ALS patients had approximately twice as much galectin-3 as normal and disease controls. These results provide the proof of principle that biomarker identification in relevant and well-controlled animal models can be translated to human disease. The challenge is to validate our biomarker candidate proteins as true biomarkers for ALS that will be useful for diagnosis and/or monitoring disease activity in future clinical trials.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Biomarcadores/análise , Galectina 3/análise , Proteômica/métodos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Biomarcadores/líquido cefalorraquidiano , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Galectina 3/líquido cefalorraquidiano , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Although changes in protein expression in spinal cord injury (SCI) would be of pivotal interest, information so far is limited. It was therefore the aim of the study to determine protein levels and post-translational modifications in the early phase following SCI in the rat. SCI was induced in Sprague-Dawley rats and sham operated rats served as controls. A gel-based proteomic approach using two-dimensional gel electrophoresis followed by quantification with specific software and subsequent identification of differentially expressed proteins by nano-ESI-LC-MS/MS was applied. Proteins of several pathways and cascades were dysregulated in SCI: 14-3-3 epsilon protein, dynein light chain 1, and tubulin beta-5 chain showed higher levels in SCI, whereas adenylyl cyclase associated protein 1, dihydropyrimidinase-related protein 2, F-actin capping protein subunit beta, glyceraldehyde-3-phosphate dehydrogenase, stress-induced phosphoprotein 1 and transthyretin showed lower levels in the injured tissue. Post-translational modifications indicated free oxygen radical attack on proteins in SCI. The occurrence of stress is indicated by deranged stress-induced phosphoprotein 1 and signaling abnormalities are reflected by adenylyl cyclase-associated protein 1 and 14-3-3 epsilon protein. The findings propose the involvement of the corresponding cascades and challenge further work into aberrant signaling and oxidative stress in SCI, which may form the basis for experimental intervention for spinal cord trauma.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/análise , Proteômica/métodos , Traumatismos da Medula Espinal/metabolismo , Proteínas 14-3-3/análise , Proteínas 14-3-3/metabolismo , Animais , Western Blotting , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Eletroforese em Gel Bidimensional , Masculino , Espectrometria de Massas , Fosforilação , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Tubulina (Proteína)/análise , Tubulina (Proteína)/metabolismoRESUMO
Systematic protein expression studies in the brain of exercising and sedentary animals have not been carried out so far and it was therefore decided to determine differences in metabolic protein levels in rat hippocampus of sedentary, voluntary and involuntary exercising rats by a proteomic approach. Aged, male Sprague-Dawley rats, 23 months old, were used for the study: the first group consisted of sedentary rats, the second of rats with voluntary exercise from five to 23 months and the third group was performing involuntary exercise on a treadmill from five to 23 months. Two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of spots followed by quantification of spots was carried out. Identification of significantly differential proteins was validated by the determination of the corresponding enzyme activity. Five individual metabolic proteins showed differential protein levels in the three groups: mitochondrial precursors of ornithine aminotransferase, isocitrate dehydrogenase [NAD] subunit alpha, malate dehydrogenase, ubiquinol-cytochrome-c reductase complex core protein 1, and ubiquitin carboxyl-terminal hydrolase isozyme L1. The unambiguously identified metabolic proteins were mainly of mitochondrial localization and fit the expectations of altered mitochondrial activity in exercise. Reduced ubiquitin carboxyl-terminal hydrolase isoenzyme L1 levels in treadmill (forced) exercise show the involvement of the proteasomal pathway as a novel finding. These results not only form the basis for functional studies elucidating mechanisms and differences between voluntary and forced exercise in hippocampal metabolism but also highlight the most intriguing aspect that exercise is affecting the brain at the protein level.
Assuntos
Hipocampo/metabolismo , Atividade Motora/fisiologia , Movimento/fisiologia , Condicionamento Físico Animal/métodos , Proteínas/metabolismo , Animais , Metabolismo Energético/fisiologia , Ativação Enzimática/fisiologia , Enzimas/metabolismo , Teste de Esforço , Hipocampo/enzimologia , Locomoção/fisiologia , Masculino , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Neurônios/enzimologia , Ratos , Regulação para Cima/fisiologia , Volição/fisiologiaRESUMO
Identification of proteins with enzymatic activity by mass spectrometry (MS) and concomitant determination of function by screening enzyme activity from two-dimensional gel electrophoresis (2DE) is one of the challenges of gel-based proteomics. In this protocol, proteins are extracted from spinal cord tissue followed by 2DE with in-gel digestion and identification by matrix-assisted laser desorption/ionization. Protein spots identified as possible enzyme of interest are punched, eluted by SDS-containing Tris buffer and renatured by buffers under reductive conditions. Enzyme activity is determined using micromethods. Within about 4 weeks, a structural and functional map can be generated and MS identification can be validated, complementing immunochemical methods. 2DE separation can be seen as a prepurification step and therefore allows activity assays from minute amounts of protein as provided in a 2DE gel spot; the method may be an alternative to the time-consuming use of recombinant enzyme techniques.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Enzimas/química , Proteômica/métodos , Animais , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Medula Espinal/enzimologiaAssuntos
Espectrometria de Massas/métodos , Proteínas/análise , Proteínas/química , Projetos de Pesquisa , Animais , Bases de Dados de Proteínas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas/instrumentação , Espectrometria de Massas/normas , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Proteínas/metabolismoRESUMO
Protein profiling in five individual mouse strains showed strain-specific expression of three hypothetical proteins (HPs). As functional and structural assignment of HPs were based on predictions and low identity to known structures, HPs were identified by MALDI-TOF/TOF, and their proposed tentative function was determined by enzyme assays. Three identified HPs were extracted from gels and renatured, and pyridoxal phosphate phosphatase, inorganic pyrophosphate phosphatase, and antioxidant activities were revealed, findings in agreement with functional predictions.
Assuntos
Hipocampo/química , Proteínas do Tecido Nervoso/análise , Sequência de Aminoácidos , Animais , Eletroforese em Gel Bidimensional/métodos , Espectrometria de Massas , Camundongos , Modelos Moleculares , Modelos Teóricos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The Harderian gland is an orbital gland located behind the ocular bulb in most terrestrial vertebrates probably functioning for production of lipid secretion to protect the eye. We herein present a protein reference database of the rat Harderian gland that may serve as analytical tool for future proteomic work, report lipid and porphyrin handling cascades, address sequence conflicts and report structures that have not been so far described by proteomics methods.
Assuntos
Glândula de Harder/metabolismo , Proteoma/análise , Ratos/anatomia & histologia , Sequência de Aminoácidos , Animais , Bases de Dados de Proteínas , Imuno-Histoquímica , Metabolismo dos Lipídeos , Lipoproteínas/química , Masculino , Dados de Sequência Molecular , Mapeamento de Peptídeos , Porfirinas/química , Porfirinas/metabolismo , Proteômica/métodos , Ratos/metabolismo , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Uroporfirinogênio Descarboxilase/químicaRESUMO
The assembly of trans-acting proteins on sequence-specific DNA cis-elements is crucial in the regulation of eukaryotic gene expression. Far upstream element binding proteins (FAB) are proteins that regulate the expression of the c-myc oncogene by binding to the far upstream element of the c-myc gene. The present study unambiguously identified the two human variants of FAB (FAB1, FAB2) in the medulloblastoma DAOY cell line and characterized their structure for the first time by tandem mass spectrometry independent of antibody availability and specificity. The study also tentatively assigned the third variant (FAB3) at the level of mass spectrometry, although tandem mass spectrometric analysis failed to corroborate the result. These findings open up an exciting possibility for discerning the cellular roles of FAB in tumor biology.
Assuntos
Neoplasias Cerebelares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meduloblastoma/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Neoplasias Cerebelares/química , Neoplasias Cerebelares/patologia , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Eletroforese em Gel Bidimensional , Humanos , Meduloblastoma/química , Meduloblastoma/patologia , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas de Ligação a RNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de TranscriçãoRESUMO
Failure in regulation of genes involved in growth and division of cells may result in pathological conditions, particularly cancer. Regulation is exerted at various levels of the transcriptional and post-transcriptional processes involving mainly nucleic acid-binding proteins. Here, we systematically explored the proteome of ten different cell lines in search for proteins potentially serving as molecular markers and/or targets for monitoring prognostic outcome and clinical therapies. High-throughput analysis, two-dimensional electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry, identified 72 nucleotide-binding proteins and their interacting partners, which were differentially expressed in the cell lines investigated. Out of the 72 identified proteins, 33 of them were specifically expressed in a single cell line (for e.g., replication protein A 32 kDa, transcription intermediary factor 1, heterogeneous ribonucleoproteins). Moreover, tumor-related proteins including breast carcinoma amplified sequence 2, zinc finger proteins, chromobox protein homologs were identified in individual cell lines. The present findings demonstrate that rich protein information can be obtained by means of proteomic analysis for better understanding of oncogenesis and pathogenesis in a global way, which in turn represents the basis for the rational designs of diagnostic and therapeutic methods.
Assuntos
Perfilação da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias/genética , Proteômica , Biomarcadores Tumorais , Humanos , Neoplasias/patologia , Ácidos Nucleicos/metabolismo , Prognóstico , Células Tumorais CultivadasRESUMO
A large part of mammalian proteomes is represented by hypothetical proteins (HP), i.e. proteins predicted from nucleic acid sequences only and protein sequences with unknown function. Databases are far from being complete and errors are expected. The legion of HP is awaiting experiments to show their existence at the protein level and subsequent bioinformatic handling in order to assign proteins a tentative function is mandatory. Two-dimensional gel-electrophoresis with subsequent mass spectrometrical identification of protein spots is an appropriate tool to search for HP in the high-throughput mode. Spots are identified by MS or by MS/MS measurements (MALDI-TOF, MALDI-TOF-TOF) and subsequent software as e.g. Mascot or ProFound. In many cases proteins can thus be unambiguously identified and characterised; if this is not the case, de novo sequencing or Q-TOF analysis is warranted. If the protein is not identified, the sequence is being sent to databases for BLAST searches to determine identities/similarities or homologies to known proteins. If no significant identity to known structures is observed, the protein sequence is examined for the presence of functional domains (databases PROSITE, PRINTS, InterPro, ProDom, Pfam and SMART), subjected to searches for motifs (ELM) and finally protein-protein interaction databases (InterWeaver, STRING) are consulted or predictions from conformations are performed. We here provide information about hypothetical proteins in terms of protein chemical analysis, independent of antibody availability and specificity and bioinformatic handling to contribute to the extension/completion of protein databases and include original work on HP in the brain to illustrate the processes of HP identification and functional assignment.
Assuntos
Bases de Dados de Proteínas , Perfilação da Expressão Gênica/métodos , Espectrometria de Massas/métodos , Publicações Periódicas como Assunto , Proteínas/química , Proteínas/metabolismo , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Animais , Medicina Baseada em Evidências , Humanos , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas/métodos , Proteínas/análise , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
The search for new structures in tumors by genomics and proteomics methods is a major goal in tumor biology and may lead to the detection of markers or antigens for the generation of tumor vaccines. The aim of this study was to identify proteins that have been predicted so far based upon their nucleic acid sequence only or show poor identity to known proteins in tumor cell lines. Cell lines of neuroblastoma, colorectal, cervix carcinoma, adenocarcinoma of the ovary, lung and breast cancer, promyelocytic leukaemia, rhabdomyosarcoma, osteosarcoma and malignant melanoma were used. Cell lysates were run on 2D gel electrophoresis with subsequent in-gel digestion and MALDI-TOF-TOF analysis. A series of 10 hypothetical proteins (HPs) were observed and three of these proteins, hypothetical protein (Q9BTE6), CGI-83 protein (Q9Y392) and similar to CG11334 (Q9BV20), were so far described in tumors exclusively. The other seven proteins were already detected at the transcriptional level in normal and tumor cell lines or tissues. In conclusion, the three HPs observed in lung cancer and malignant melanoma may be candidates for development of tumor markers and generation of tumor vaccines.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Neoplasias/análise , Proteômica , Sequência de Aminoácidos , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/genéticaRESUMO
BACKGROUND: Chaperones (CH) play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression.Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. RESULTS: A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. CONCLUSIONS: The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity.