RESUMO
Reversible protein phosphorylation on serine, threonine, and tyrosine residues is essential for fast, specific, and accurate signal transduction in cells. Up to now, the identification and quantification of phosphorylated amino acids, peptides, and proteins continue to be one of the significant challenges in contemporary bioanalytical research. In this paper, a series of surface grafted monoliths in the capillary format targeting phosphorylated serine has been prepared by first synthesizing a monolithic core substrate material based on trimethylolpropane trimethacrylate, onto which a thin surface-imprinted layer was established by oriented photografting of a variety of mono- and bis-imidazolium host monomers at subzero temperature, using six different continuous or pulsed UV light sources. The imprinted monolith capillaries were evaluated in a capillary liquid chromatographic system connected to a mass spectrometer in order to test the specific retention of phosphorylated peptides. Site-specific recognition selectivity and specificity for phosphorylated serine was demonstrated when separating amino acids and peptides, proving that the optimized materials could be used as novel trapping media in affinity-based phosphoproteomic analysis.
Assuntos
Angiotensina II/metabolismo , Cromatografia de Afinidade/métodos , Imidazóis/química , Impressão Molecular/métodos , Fosfopeptídeos/isolamento & purificação , Polímeros/química , Raios Ultravioleta , Angiotensina II/química , Humanos , Fosfopeptídeos/química , Fosforilação , Polímeros/efeitos da radiaçãoRESUMO
There is increasing evidence that multiple proteins involved in key regulatory processes in mitochondria are phosphorylated in mammalian tissues. Insulin regulates glucose metabolism by phosphorylation-dependent signaling and has been shown to stimulate ATP synthesis in human skeletal muscle. Here, we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO(2) phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included the majority of novel sites. Phosphorylation sites detected more often or exclusively in insulin-stimulated samples include multiple sites in mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid metabolism, as well as several components of the newly defined mitochondrial inner membrane organizing system (MINOS). In conclusion, the present study demonstrates that insulin increases the phosphorylation of several mitochondrial proteins in human skeletal muscle in vivo and provides a first step in the understanding of how insulin potentially regulates mitochondrial processes by phosphorylation-dependent mechanisms.
Assuntos
Insulina/farmacologia , Mitocôndrias Musculares/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/efeitos dos fármacos , Adulto , Sítios de Ligação , Cromatografia Líquida , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Insulina/administração & dosagem , Sistemas de Infusão de Insulina , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/classificação , Músculo Esquelético/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Fosfopeptídeos/classificação , Fosfopeptídeos/metabolismo , Fosfoproteínas/classificação , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
We have studied the role(s) of maturation drying in the acquisition of germinability, seedling vigor and pathogen resistance by comparing the proteome changes in maize embryo and endosperm during mature and prematurely imposed drying. Prematurely imposed dried seeds at 40 days after pollination (DAP) germinated almost as well as mature seeds (at 65 DAP), but their seedling growth was slower and they were seriously infected by fungi. A total of 80 and 114 proteins were identified to change at least two-fold (p < 0.05) in abundance during maturation drying in embryo and endosperm, respectively. Fewer proteins (48 and 59 in embryo and endosperm, respectively) changed in abundance during prematurely imposed drying. A number of proteins, 33 and 38 in embryo and endosperm, respectively, changed similarly in abundance during both maturation and prematurely imposed drying. Storage proteins were abundant in this group and may contribute to the acquisition of seed germinability. However, a relatively large number of proteins changed in the embryo (47 spots) and endosperm (76 spots) specifically during maturation drying. Among these proteins, storage proteins in the embryo and defense proteins in the endosperm may be particularly important for seedling vigor and resistance to fungal infection, respectively.
Assuntos
Fungos/patogenicidade , Germinação , Proteínas de Plantas/metabolismo , Proteômica , Sementes/metabolismo , Zea mays/embriologia , Eletroforese em Gel Bidimensional , Sementes/fisiologia , Zea mays/microbiologia , Zea mays/fisiologiaRESUMO
Susceptibility to stress plays a crucial role in the development of psychiatric disorders such as unipolar depression and post-traumatic stress disorder. In the present study the chronic mild stress rat model of depression was used to reveal stress-susceptible and stress-resilient rats. Large-scale proteomics was used to map hippocampal protein alterations in different stress states. Membrane proteins were successfully captured by two-phase separation and peptide based proteomics. Using iTRAQ labeling coupled with mass spectrometry, more than 2000 proteins were quantified and 73 proteins were found to be differentially expressed. Stress susceptibility was associated with increased expression of a sodium-channel protein (SCN9A) currently investigated as a potential antidepressant target. Differential protein profiling also indicated stress susceptibility to be associated with deficits in synaptic vesicle release involving SNCA, SYN-1, and AP-3. Our results indicate that increased oxidative phosphorylation (COX5A, NDUFB7, NDUFS8, COX5B, and UQCRB) within the hippocampal CA regions is part of a stress-protection mechanism.
Assuntos
Adaptação Biológica/genética , Transtorno Depressivo/metabolismo , Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Transtorno Depressivo/etiologia , Transtorno Depressivo/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Masculino , Espectrometria de Massas , Fosforilação Oxidativa , Estimulação Luminosa/efeitos adversos , Proteômica , Ratos , Ratos Wistar , Estresse Fisiológico , Sacarose/administração & dosagem , Sinapsinas/genética , Sinapsinas/metabolismo , Vesículas Sinápticas/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Large-scale quantitative analyses of biological systems are often performed with few replicate experiments, leading to multiple nonidentical data sets due to missing values. For example, mass spectrometry driven proteomics experiments are frequently performed with few biological or technical replicates due to sample-scarcity or due to duty-cycle or sensitivity constraints, or limited capacity of the available instrumentation, leading to incomplete results where detection of significant feature changes becomes a challenge. This problem is further exacerbated for the detection of significant changes on the peptide level, for example, in phospho-proteomics experiments. In order to assess the extent of this problem and the implications for large-scale proteome analysis, we investigated and optimized the performance of three statistical approaches by using simulated and experimental data sets with varying numbers of missing values. We applied three tools, including standard t test, moderated t test, also known as limma, and rank products for the detection of significantly changing features in simulated and experimental proteomics data sets with missing values. The rank product method was improved to work with data sets containing missing values. Extensive analysis of simulated and experimental data sets revealed that the performance of the statistical analysis tools depended on simple properties of the data sets. High-confidence results were obtained by using the limma and rank products methods for analyses of triplicate data sets that exhibited more than 1000 features and more than 50% missing values. The maximum number of differentially represented features was identified by using limma and rank products methods in a complementary manner. We therefore recommend combined usage of these methods as a novel and optimal way to detect significantly changing features in these data sets. This approach is suitable for large quantitative data sets from stable isotope labeling and mass spectrometry experiments and should be applicable to large data sets of any type. An R script that implements the improved rank products algorithm and the combined analysis is available.
Assuntos
Interpretação Estatística de Dados , Proteoma/metabolismo , Software , Simulação por Computador , Proteoma/química , Proteômica , Melhoria de Qualidade , Reprodutibilidade dos Testes , Projetos de Pesquisa , Razão Sinal-RuídoRESUMO
Phosphorylation of mitochondrial proteins in a variety of biological processes is increasingly being recognized and may contribute to the differences in function and energy demands observed in mitochondria from different tissues such as liver, heart, and skeletal muscle. Here, we used a combination of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS on isolated mitochondria to investigate the tissue-specific mitochondrial phosphoproteomes of rat liver, heart, and skeletal muscle. In total, we identified 899 phosphorylation sites in 354 different mitochondrial proteins including 479 potential novel sites. Most phosphorylation sites were detected in liver mitochondria (594), followed by heart (448) and skeletal muscle (336), and more phosphorylation sites were exclusively identified in liver mitochondria than in heart and skeletal muscle. Bioinformatics analysis pointed out enrichment for phosphoproteins involved in amino acid and fatty acid metabolism in liver mitochondria, whereas heart and skeletal muscle were enriched for phosphoproteins involved in energy metabolism, in particular, tricarboxylic acid cycle and oxidative phosphorylation. Multiple tissue-specific phosphorylation sites were identified in tissue-specific enzymes such as those encoded by HMGCS2, BDH1, PCK2, CPS1, and OTC in liver mitochondria, and CKMT2 and CPT1B in heart and skeletal muscle. Kinase prediction showed an important role for PKA and PKC in all tissues but also for proline-directed kinases in liver mitochondria. In conclusion, we provide a comprehensive map of mitochondrial phosphorylation sites, which covers approximately one-third of the mitochondrial proteome and can be targeted for the investigation of tissue-specific regulation of mitochondrial biological processes.
Assuntos
Fígado/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Masculino , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/química , Dados de Sequência Molecular , Especificidade de Órgãos , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. RESULTS: From 150 µg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. CONCLUSIONS: Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.
Assuntos
Proteína Morfogenética Óssea 2/genética , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Fosfoproteínas/genética , Pele/metabolismo , Animais , Proteína Morfogenética Óssea 2/metabolismo , Caseína Quinases/genética , Caseína Quinases/metabolismo , Diferenciação Celular , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Espectrometria de Massas , Células-Tronco Mesenquimais/citologia , Camundongos , Osteoblastos/citologia , Fosfoproteínas/metabolismo , Fosforilação , Transdução de Sinais , Pele/citologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Recently, bryophytes, which diverged from the ancestor of seed plants more than 400 million years ago, came into focus in photosynthesis research as they can provide valuable insights into the evolution of photosynthetic complexes during the adaptation to terrestrial life. This study isolated intact photosystem I (PSI) with its associated light-harvesting complex (LHCI) from the moss Physcomitrella patens and characterized its structure, polypeptide composition, and light-harvesting function using electron microscopy, mass spectrometry, biochemical, and physiological methods. It became evident that Physcomitrella possesses a strikingly high number of isoforms for the different PSI core subunits as well as LHCI proteins. It was demonstrated that all these different subunit isoforms are expressed at the protein level and are incorporated into functional PSI-LHCI complexes. Furthermore, in contrast to previous reports, it was demonstrated that Physcomitrella assembles a light-harvesting complex consisting of four light-harvesting proteins forming a higher-plant-like PSI superstructure.
Assuntos
Bryopsida/metabolismo , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Bryopsida/química , Bryopsida/genética , Bryopsida/efeitos da radiação , Luz , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema I/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.
Assuntos
Mitocôndrias Musculares/metabolismo , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Descanso/fisiologia , Adulto , Ciclo do Ácido Cítrico , Transporte de Elétrons , Humanos , Pessoa de Meia-Idade , Fosforilação , Proteínas Quinases/metabolismoRESUMO
The development of metastasis is a complex, multistep process that remains poorly defined. To identify proteins involved in the colonization phase of the metastatic process, we compared the proteome of tumors derived from inoculation of a panel of isogenic human cancer cell lines with different metastatic capabilities into the mammary fat pad of immunodeficient mice. Using a protein standard generated by SILAC-labeling, a total of 675 proteins were identified and 30 were differentially expressed between at least two of the tumors. The protein standard contained the proteomes of seven cell lines from multiple histogenic origins and displayed superior features compared to standard super-SILAC. The expression of some proteins correlated with metastatic capabilities, such as myosin-9 (nonmuscle myosin II A) and L-lactate dehydrogenase A, while the expression of elongation factor tu correlated inversely to metastatic capabilities. The expression of these proteins was biochemically validated, and expression of myosin-9 in clinical breast cancer samples was further shown to be altered in primary tumors versus corresponding lymph node metastasis. Our study demonstrates an improved strategy for quantitative comparison of an unlimited number of tumor tissues, and provides novel insights into key proteins associated with the colonization phase of metastasis formation.
Assuntos
Regulação Neoplásica da Expressão Gênica , Marcação por Isótopo/métodos , Metástase Neoplásica/genética , Neoplasias/genética , Proteínas/genética , Proteômica/métodos , Animais , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos SCID , Miosinas/análise , Miosinas/genética , Metástase Neoplásica/patologia , Neoplasias/patologia , Proteínas/análise , Espectrometria de Massas em Tandem/métodosRESUMO
Regulation of transcription requires cooperation between sequence-specific transcription factors and numerous coregulatory proteins. In IL-4/IL-13 signaling several coactivators for STAT6 have been identified, but the molecular mechanisms of STAT6-mediated gene transcription are still not fully understood. Here we identified by proteomic approach that the PTB-associated splicing factor (PSF) interacts with STAT6. In intact cells the interaction was observed only after IL-4 stimulation. The IL-4-induced tyrosine phosphorylation of both STAT6 and PSF is a prerequisite for the efficient association of the two proteins. Functional analysis demonstrated that ectopic expression of PSF resulted in inhibition of STAT6-mediated transcriptional activation and mRNA expression of the Igε germline heavy chain gene, whereas knockdown of PSF increased the STAT6-mediated responses. PSF recruited histone deacetylase 1 (HDAC1) to the STAT6 transcription complex, which resulted in reduction of H3 acetylation at the promoter regions of Ig heavy chain germline Igε and inhibition of STAT6-mediated transcription. In addition, the HDACs inhibitor trichostatin A (TSA) enhanced H3 acetylation, and reverted the PSF-mediated transcriptional repression of Igε gene transcription. In summary, these results identify PSF as a repressor of STAT6-mediated transcription that functions through recruitment of HDAC to the STAT6 transcription complex, and delineates a novel regulatory mechanism of IL-4 signaling that may have implications in the pathogenesis of allergic diseases and pharmacological HDAC inhibition in lymphomas.
Assuntos
Histona Desacetilase 1/metabolismo , Cadeias épsilon de Imunoglobulina/genética , Proteínas de Ligação a RNA/fisiologia , Fator de Transcrição STAT6/fisiologia , Transcrição Gênica , Genes de Imunoglobulinas , Células HeLa , Humanos , Interleucina-4/farmacologia , Fator de Processamento Associado a PTB , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras , Ativação TranscricionalRESUMO
The transfer-messenger RNA (tmRNA)-mediated trans-translation mechanism is highly conserved in bacteria and functions primarily as a system for the rescue of stalled ribosomes and the removal of aberrantly produced proteins. Here, we show that in the antibiotic-producing soil bacterium Streptomyces coelicolor, trans-translation has a specialized role in stress management. Analysis of proteins that were carboxy-terminally His(8)-tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat-shock protein 70, thiostrepton-induced protein A, universal stress protein A, elongation factor Tu3, and the cell-cycle control proteins DasR, SsgA, SsgF and SsgR. Although tmRNA-tagged proteins are degraded swiftly, the translation of dnaK and dasR messenger RNAs (mRNAs) depends fully on tmRNA, whereas transcription is unaffected. The data unveil a surprisingly dedicated functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/fisiologia , RNA de Transferência/fisiologia , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Modelos Biológicos , Proteoma/análise , Proteoma/metabolismo , Streptomyces coelicolor/genéticaRESUMO
Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine 27 (K27), and it is believed that this activity mediates transcriptional repression. Despite the recent progress in understanding PcG function, the molecular mechanisms by which the PcG proteins repress transcription, as well as the mechanisms that lead to the activation of PcG target genes are poorly understood. To gain insight into these mechanisms, we have determined the global changes in histone modifications in embryonic stem (ES) cells lacking the PcG protein Suz12 that is essential for PRC2 activity. We show that loss of PRC2 activity results in a global increase in H3K27 acetylation. The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and CBP. Based on these data, we propose that the PcG proteins in part repress transcription by preventing the binding of acetyltransferases to PcG target genes.
Assuntos
Regulação da Expressão Gênica , Histonas/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Acetilação , Animais , Células-Tronco Embrionárias/metabolismo , Técnicas de Inativação de Genes , Histona Acetiltransferases/metabolismo , Histonas/química , Lisina/metabolismo , Metilação , Camundongos , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Proteínas Repressoras/genéticaRESUMO
Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one of the bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized phosphorylation motif "x(pT)xEx".
Assuntos
Proteínas de Bactérias/química , Fosfoproteínas/análise , Software , Streptomyces coelicolor/química , Streptomyces coelicolor/crescimento & desenvolvimento , Motivos de Aminoácidos , Biologia Computacional , Precipitação Fracionada/métodos , Fosfoproteínas/química , Fosforilação , Filogenia , Proteínas Quinases/química , Proteínas Quinases/classificação , Elementos Reguladores de Transcrição , Serina/química , Esporos Bacterianos/química , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces coelicolor/classificação , Streptomyces coelicolor/genética , Treonina/química , Tirosina/químicaRESUMO
MOTIVATION: Fuzzy c-means clustering is widely used to identify cluster structures in high-dimensional datasets, such as those obtained in DNA microarray and quantitative proteomics experiments. One of its main limitations is the lack of a computationally fast method to set optimal values of algorithm parameters. Wrong parameter values may either lead to the inclusion of purely random fluctuations in the results or ignore potentially important data. The optimal solution has parameter values for which the clustering does not yield any results for a purely random dataset but which detects cluster formation with maximum resolution on the edge of randomness. RESULTS: Estimation of the optimal parameter values is achieved by evaluation of the results of the clustering procedure applied to randomized datasets. In this case, the optimal value of the fuzzifier follows common rules that depend only on the main properties of the dataset. Taking the dimension of the set and the number of objects as input values instead of evaluating the entire dataset allows us to propose a functional relationship determining the fuzzifier directly. This result speaks strongly against using a predefined fuzzifier as typically done in many previous studies. Validation indices are generally used for the estimation of the optimal number of clusters. A comparison shows that the minimum distance between the centroids provides results that are at least equivalent or better than those obtained by other computationally more expensive indices.
Assuntos
Análise por Conglomerados , Lógica Fuzzy , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reconhecimento Automatizado de Padrão/métodos , ProteômicaRESUMO
Genetic studies have repeatedly shown that the Bromodomain containing 1 gene, BRD1, is involved in determining mental health, and the importance of the BRD1 protein for normal brain function has been studied in both cell models and constitutive haploinsufficient Brd1+/- mice. Homozygosity for inactivated Brd1 alleles is lethal during embryonic development in mice. In order to further characterize the molecular functions of BRD1 in the brain, we have developed a novel Brd1 knockout mouse model (Brd1-/-) with bi-allelic conditional inactivation of Brd1 in the central nervous system. Brd1-/- mice were viable but smaller and with reduced muscle strength. They showed reduced exploratory behavior and increased sensitivity to pentylenetetrazole-induced seizures supporting the previously described GABAergic dysfunction in constitutive Brd1+/- mice. Because BRD1 takes part in protein complexes with histone binding and modifying functions, we investigated the effect of BRD1 depletion on the global histone modification pattern in mouse brain by mass spectrometry. We found decreased levels of histone H3 acetylation (H3K9ac, H3K14ac, and H3K18ac) and increased N-tail clipping in consequence of BRD1 depletion. Collectively, the presented results support that BRD1 controls gene expression at the epigenetic level by regulating histone H3 proteoforms in the brain.
Assuntos
Encéfalo/metabolismo , Histona Acetiltransferases/genética , Histonas/metabolismo , Esquizofrenia/genética , Convulsões/genética , Acetilação , Animais , Histona Acetiltransferases/metabolismo , Histonas/genética , Camundongos , Camundongos Knockout , Esquizofrenia/metabolismo , Convulsões/metabolismoRESUMO
OBJECTIVE: Increasing adaptive thermogenesis by stimulating browning in white adipose tissue is a promising method of improving metabolic health. However, the molecular mechanisms underlying this transition remain elusive. Our study examined the molecular determinants driving the differentiation of precursor cells into thermogenic adipocytes. METHODS: In this study, we conducted temporal high-resolution proteomic analysis of subcutaneous white adipose tissue (scWAT) after cold exposure in mice. This was followed by loss- and gain-of-function experiments using siRNA-mediated knockdown and CRISPRa-mediated induction of gene expression, respectively, to evaluate the function of the transcriptional regulator Y box-binding protein 1 (YBX1) during adipogenesis of brown pre-adipocytes and mesenchymal stem cells. Transcriptomic analysis of mesenchymal stem cells following induction of endogenous Ybx1 expression was conducted to elucidate transcriptomic events controlled by YBX1 during adipogenesis. RESULTS: Our proteomics analysis uncovered 509 proteins differentially regulated by cold in a time-dependent manner. Overall, 44 transcriptional regulators were acutely upregulated following cold exposure, among which included the cold-shock domain containing protein YBX1, peaking after 24 h. Cold-induced upregulation of YBX1 also occurred in brown adipose tissue, but not in visceral white adipose tissue, suggesting a role of YBX1 in thermogenesis. This role was confirmed by Ybx1 knockdown in brown and brite preadipocytes, which significantly impaired their thermogenic potential. Conversely, inducing Ybx1 expression in mesenchymal stem cells during adipogenesis promoted browning concurrent with an increased expression of thermogenic markers and enhanced mitochondrial respiration. At a molecular level, our transcriptomic analysis showed that YBX1 regulates a subset of genes, including the histone H3K9 demethylase Jmjd1c, to promote thermogenic adipocyte differentiation. CONCLUSION: Our study mapped the dynamic proteomic changes of murine scWAT during browning and identified YBX1 as a novel factor coordinating the genomic mechanisms by which preadipocytes commit to brite/beige lineage.
Assuntos
Tecido Adiposo Branco/metabolismo , Termogênese/genética , Termogênese/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adipócitos Marrons/metabolismo , Adipogenia , Tecido Adiposo Marrom/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Proteômica , Gordura Subcutânea/metabolismo , Transcriptoma , Regulação para CimaRESUMO
Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)-nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile-water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)-NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 microg of mouse sample or 50 microg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC-IMAC and LC-MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC-IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects.
Assuntos
Extratos Celulares/química , Cromatografia de Afinidade/métodos , Misturas Complexas/química , Compostos Férricos/química , Ácido Nitrilotriacético/análogos & derivados , Fosfopeptídeos/química , Acetonitrilas , Animais , Bovinos , Bases de Dados de Proteínas , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Concentração de Íons de Hidrogênio , Linfócitos/química , Camundongos , Ácido Nitrilotriacético/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfopeptídeos/metabolismo , Fosfoproteínas , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismoRESUMO
Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC gamma subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.