RESUMO
Hormones and neurotransmitters are essential to homeostasis, and their disruptions are connected to diseases ranging from cancer to anxiety. The differential reactivation of endobiotic glucuronides by gut microbial ß-glucuronidase (GUS) enzymes may influence interindividual differences in the onset and treatment of disease. Using multi-omic, in vitro, and in vivo approaches, we show that germ-free mice have reduced levels of active endobiotics and that distinct gut microbial Loop 1 and FMN GUS enzymes drive hormone and neurotransmitter reactivation. We demonstrate that a range of FDA-approved drugs prevent this reactivation by intercepting the catalytic cycle of the enzymes in a conserved fashion. Finally, we find that inhibiting GUS in conventional mice reduces free serotonin and increases its inactive glucuronide in the serum and intestines. Our results illuminate the indispensability of gut microbial enzymes in sustaining endobiotic homeostasis and indicate that therapeutic disruptions of this metabolism promote interindividual response variabilities.
Assuntos
Microbioma Gastrointestinal , Glucuronidase , Homeostase , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Camundongos , Glucuronidase/metabolismo , Camundongos Endogâmicos C57BL , Serotonina/metabolismo , Glucuronídeos/metabolismo , Humanos , Intestinos/microbiologia , Masculino , Vida Livre de GermesRESUMO
Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth of protein quantification, especially for proteins and modifications of biological interest. Here, to simultaneously advance all these aims, we developed prioritized Single-Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands of prioritized peptides across all single cells (thus increasing data completeness) while maximizing instrument time spent analyzing identifiable peptides, thus increasing proteome depth. These strategies increased the sensitivity, data completeness and proteome coverage over twofold. The gains enabled quantifying protein variation in untreated and lipopolysaccharide-treated primary macrophages. Within each condition, proteins covaried within functional sets, including phagosome maturation and proton transport, similarly across both treatment conditions. This covariation is coupled to phenotypic variability in endocytic activity. pSCoPE also enabled quantifying proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is freely available and widely applicable, especially for analyzing proteins of interest without sacrificing proteome coverage. Support for pSCoPE is available at http://scp.slavovlab.net/pSCoPE .
Assuntos
Proteoma , Proteômica , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas , Peptídeos/química , MacrófagosRESUMO
Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.
Assuntos
Peptídeos/isolamento & purificação , Proteoma/isolamento & purificação , Proteômica/métodos , Análise de Célula Única/métodos , Animais , Benchmarking , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Células HeLa , Humanos , Indicadores e Reagentes/química , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Peptídeos/química , Peptídeos/classificação , Cultura Primária de Células , Proteoma/química , Proteoma/classificação , Células RAW 264.7 , Análise de Célula Única/normas , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normas , Células U937RESUMO
BACKGROUND: Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis. RESULTS: To overcome this limitation, we develop SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. Parallel measurements of transcripts by 10× Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus, SCoPE2 supports quantification with improved count statistics. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. CONCLUSIONS: Even in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.
Assuntos
Macrófagos/metabolismo , Proteômica , Transcriptoma , Linhagem Celular , Citocinas/genética , Proteínas Alimentares , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ativação de Macrófagos , Monócitos/metabolismo , Fenótipo , Proteoma/genéticaRESUMO
Lower glycolysis involves a series of reversible reactions, which interconvert intermediates that also feed anabolic pathways. 3-phosphoglycerate (3-PG) is an abundant lower glycolytic intermediate that feeds serine biosynthesis via the enzyme phosphoglycerate dehydrogenase, which is genomically amplified in several cancers. Phosphoglycerate mutase 1 (PGAM1) catalyzes the isomerization of 3-PG into the downstream glycolytic intermediate 2-phosphoglycerate (2-PG). PGAM1 needs to be histidine phosphorylated to become catalytically active. We show that the primary PGAM1 histidine phosphate donor is 2,3-bisphosphoglycerate (2,3-BPG), which is made from the glycolytic intermediate 1,3-bisphosphoglycerate (1,3-BPG) by bisphosphoglycerate mutase (BPGM). When BPGM is knocked out, 1,3-BPG can directly phosphorylate PGAM1. In this case, PGAM1 phosphorylation and activity are decreased, but nevertheless sufficient to maintain normal glycolytic flux and cellular growth rate. 3-PG, however, accumulates, leading to increased serine synthesis. Thus, one biological function of BPGM is controlling glycolytic intermediate levels and thereby serine biosynthetic flux.
Assuntos
Ácidos Glicéricos/metabolismo , Fosfoglicerato Mutase/metabolismo , Serina/metabolismo , Humanos , Fosfoglicerato Mutase/deficiência , Células Tumorais CultivadasRESUMO
When cells mobilize nutrients from protein, they generate a fingerprint of peptide fragments that reflects the net action of proteases and the identities of the affected proteins. Analyzing these mixtures falls into a grey area between proteomics and metabolomics that is poorly served by existing technology. Herein, we describe an emerging digestomics strategy that bridges this gap and allows mixtures of proteolytic fragments to be quantitatively mapped with an amino acid level of resolution. We describe recent successes using this technique, including a case where digestomics provided the link between hemoglobin digestion by the malaria parasite and the world-wide distribution of chloroquine resistance. We highlight other areas of microbiology and cancer research that are well-suited to this emerging technology.
Assuntos
Hemoglobinas/metabolismo , Malária/metabolismo , Proteômica/métodos , Proteínas de Protozoários/metabolismo , Antimaláricos/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos , Humanos , Malária/tratamento farmacológico , Malária/parasitologia , Plasmodium/efeitos dos fármacos , ProteóliseRESUMO
Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.
Assuntos
Fosfatos de Inositol/metabolismo , Redes e Vias Metabólicas/fisiologia , Polifosfatos/metabolismo , Transdução de Sinais/fisiologia , Difosfatos/metabolismo , Células Eucarióticas/metabolismo , Glucose/metabolismo , Magnésio , Nucleotídeos/metabolismo , Fosforilação , Proteoma , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
During cell division, polarized epithelial cells employ mechanisms to preserve cell polarity and tissue integrity. In dividing cells of the mammalian skin, planar cell polarity (PCP) is maintained through the bulk internalization, equal segregation, and polarized recycling of cortical PCP proteins. The dramatic redistribution of PCP proteins coincides precisely with cell-cycle progression, but the mechanisms coordinating PCP and mitosis are unknown. Here we identify Plk1 as a master regulator of PCP dynamics during mitosis. Plk1 interacts with core PCP component Celsr1 via a conserved polo-box domain (PBD)-binding motif, localizes to mitotic endosomes, and directly phosphorylates Celsr1. Plk1-dependent phosphorylation activates the endocytic motif specifically during mitosis, allowing bulk recruitment of Celsr1 into endosomes. Inhibiting Plk1 activity blocks PCP internalization and perturbs PCP asymmetry. Mimicking dileucine motif phosphorylation is sufficient to drive Celsr1 internalization during interphase. Thus, Plk1-mediated phosphorylation of Celsr1 ensures that PCP redistribution is precisely coordinated with mitotic entry.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Polaridade Celular/fisiologia , Endossomos/metabolismo , Queratinócitos/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Endocitose/fisiologia , Imunofluorescência , Células HeLa , Humanos , Interfase , Queratinócitos/citologia , Camundongos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Quinase 1 Polo-LikeRESUMO
Replication of human cytomegalovirus (HCMV) is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus-coded kinase augments the replication of HCMV by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC-labeled human fibroblasts during infection with either wild-type HCMV or a pUL97 kinase-dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high-resolution MS. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication.
Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Fibroblastos/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteoma/análise , Proteômica/métodos , Western Blotting , Células Cultivadas , Cromatografia Líquida , Infecções por Citomegalovirus/virologia , Fibroblastos/virologia , Humanos , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Espectrometria de Massas em Tandem , Replicação ViralRESUMO
UNLABELLED: Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1-17, 2014, http://dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076-1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells. IMPORTANCE: The tumor suppressor p53, a master regulator of cellular responses to stress, is inactivated and destroyed in cells infected by species C human adenoviruses, such as type 5. It is targeted for proteasomal degradation by the action of a virus-specific E3 ubiquitin ligase that contains the viral E1B 55-kDa and E4 Orf6 proteins, while the E4 Orf3 protein has been reported to block its ability to stimulate expression of p53-dependent genes. The comparisons reported here of the posttranslational modifications and activities of p53 populations that accumulate in infected normal human cells in the absence of both mechanisms of inactivation or of only the E3 ligase revealed little impact of the E4 Orf3 protein. These observations indicate that E4 Orf3-dependent disruption of Pml bodies does not have a major effect on the pattern of p53 posttranslational modifications in adenovirus-infected cells. Furthermore, they suggest that one or more additional viral proteins contribute to blocking p53 activation and the consequences that are deleterious for viral reproduction, such as apoptosis or cell cycle arrest.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Fases de Leitura Aberta , Proteína Supressora de Tumor p53/metabolismo , Adenoviridae/genética , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas E4 de Adenovirus/genética , Linhagem Celular , Humanos , Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Identification of novel genetic risk factors is imperative for a better understanding of B lymphomagenesis and for the development of novel therapeutic strategies. TRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. The present study aimed to identify novel oncogenes involved in malignant transformation of TRAF3-deficient B cells. METHODS: We used microarray analysis to identify genes differentially expressed in TRAF3-/- mouse splenic B lymphomas. We employed lentiviral vector-mediated knockdown or overexpression to manipulate gene expression in human multiple myeloma (MM) cell lines. We analyzed cell apoptosis and proliferation using flow cytometry, and performed biochemical studies to investigate signaling mechanisms. To delineate protein-protein interactions, we applied affinity purification followed by mass spectrometry-based sequencing. RESULTS: We identified mutated in colorectal cancer (MCC) as a gene strikingly up-regulated in TRAF3-deficient mouse B lymphomas and human MM cell lines. Aberrant up-regulation of MCC also occurs in a variety of primary human B cell malignancies, including non-Hodgkin lymphoma (NHL) and MM. In contrast, MCC expression was not detected in normal or premalignant TRAF3-/- B cells even after treatment with B cell stimuli, suggesting that aberrant up-regulation of MCC is specifically associated with malignant transformation of B cells. In elucidating the functional roles of MCC in malignant B cells, we found that lentiviral shRNA vector-mediated knockdown of MCC induced apoptosis and inhibited proliferation in human MM cells. Experiments of knockdown and overexpression of MCC allowed us to identify several downstream targets of MCC in human MM cells, including phospho-ERK, c-Myc, p27, cyclin B1, Mcl-1, caspases 8 and 3. Furthermore, we identified 365 proteins (including 326 novel MCC-interactors) in the MCC interactome, among which PARP1 and PHB2 were two hubs of MCC signaling pathways in human MM cells. CONCLUSIONS: Our results indicate that in sharp contrast to its tumor suppressive role in colorectal cancer, MCC functions as an oncogene in B cells. Our findings suggest that MCC may serve as a diagnostic marker and therapeutic target in B cell malignancies, including NHL and MM.
Assuntos
Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Genes MCC/genética , Oncogenes/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Imunoprecipitação da Cromatina , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Linfoma não Hodgkin/genética , Camundongos , Camundongos Knockout , Mieloma Múltiplo/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proibitinas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 3 Associado a Receptor de TNF/deficiência , Espectrometria de Massas em TandemRESUMO
Ras-driven cancer cells upregulate basal autophagy that degrades and recycles intracellular proteins and organelles. Autophagy-mediated proteome degradation provides free amino acids to support metabolism and macromolecular synthesis, which confers a survival advantage in starvation and promotes tumorigenesis. While the degradation of isolated protein substrates by autophagy has been implicated in controlling cellular function, the extent and specificity by which autophagy remodels the cellular proteome and the underlying functional consequences were unknown. Here we compared the global proteome of autophagy-functional and -deficient Ras-driven cancer cells, finding that autophagy affects the majority of the proteome yet is highly selective. While levels of vesicle trafficking proteins important for autophagy are preserved during starvation-induced autophagy, deleterious inflammatory response pathway components are eliminated even under basal conditions, preventing cytokine-induced paracrine cell death. This reveals the global, functional impact of autophagy-mediated proteome remodeling on cell survival and identifies critical autophagy substrates that mediate this process.
Assuntos
Autofagia , Imunidade Inata , Proteoma/fisiologia , Proteínas ras/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Camundongos , Transporte Proteico , Vesículas TransportadorasRESUMO
Protein histidine phosphorylation is increasingly recognized as a critical posttranslational modification (PTM) in central metabolism and cell signaling. Still, the detection of phosphohistidine (pHis) in the proteome has remained difficult due to the scarcity of tools to enrich and identify this labile PTM. To address this, we report the first global proteomic analysis of pHis proteins, combining selective immunoenrichment of pHis peptides and a bioinformatic strategy based on mechanistic insight into pHis peptide gas-phase fragmentation during LC-MS/MS. We show that collision-induced dissociation (CID) of pHis peptides produces prominent characteristic neutral losses of 98, 80, and 116 Da. Using isotopic labeling studies, we also demonstrate that the 98 Da neutral loss occurs via gas-phase phosphoryl transfer from pHis to the peptide C-terminal α-carboxylate or to Glu/Asp side chain residues if present. To exploit this property, we developed a software tool that screens LC-MS/MS spectra for potential matches to pHis-containing peptides based on their neutral loss pattern. This tool was integrated into a proteomics workflow for the identification of endogenous pHis-containing proteins in cellular lysates. As an illustration of this strategy, we analyzed pHis peptides from glycerol-fed and mannitol-fed Escherichia coli cells. We identified known and a number of previously speculative pHis sites inferred by homology, predominantly in the phosphoenolpyruvate:sugar transferase system (PTS). Furthermore, we identified two new sites of histidine phosphorylation on aldehyde-alcohol dehydrogenase (AdhE) and pyruvate kinase (PykF) enzymes, previously not known to bear this modification. This study lays the groundwork for future pHis proteomics studies in bacteria and other organisms.
Assuntos
Histidina/análogos & derivados , Fragmentos de Peptídeos/análise , Fosfopeptídeos/análise , Proteômica/métodos , Sequência de Aminoácidos , Escherichia coli/química , Proteínas de Escherichia coli/química , Histidina/análise , Dados de Sequência Molecular , Espectrometria de Massas em Tandem/métodosRESUMO
The RNA polymerase II (Pol-II) holoenzyme, responsible for messenger RNA production, typically consists of 10-12 subunits. Our laboratory previously demonstrated that maternally deposited, long, noncoding, template RNAs are essential for programmed genome rearrangements in the ciliate Oxytricha trifallax. Here we show that such RNAs are bidirectionally transcribed and transported to the zygotic nucleus. The gene encoding the second-largest subunit of Pol-II, Rpb2, has undergone gene duplication, and the two paralogs, Rpb2-a and -b, display different expression patterns. Immunoprecipitation of double-stranded RNAs identified an association with Rpb2-a. Through immunoprecipitation and mass spectrometry, we show that Rpb2-a in early zygotes appears surprisingly unassociated with other Pol II subunits. A partial loss of function of Rpb2-a leads to an increase in expression of transposons and other germline-limited satellite repeats. We propose that evolutionary divergence of the Rpb2 paralogs has led to acquisition of transcription-independent functions during sexual reproduction that may contribute to the negative regulation of germline gene expression.
Assuntos
Rearranjo Gênico/genética , Genoma , Oxytricha/enzimologia , Oxytricha/genética , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Extratos Celulares , Cromatografia Líquida , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células Germinativas/metabolismo , Espectrometria de Massas , Oxytricha/crescimento & desenvolvimento , Peptídeos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Zigoto/metabolismoRESUMO
The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues-for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated.
Assuntos
Fibroblastos/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Proteínas E1B de Adenovirus/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/metabolismo , Humanos , Metilação , Fosforilação/genética , Proteólise , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Calcium-dependent cysteine proteases of the calpain family are modulatory proteases that cleave their substrates in a limited manner. Among their substrates, calpains target vertebrate and invertebrate IκB proteins. Because proteolysis by calpains potentially generates novel protein functions, it is important to understand how this affects NFκB activity. We investigate the action of Calpain A (CalpA) on the Drosophila melanogaster IκB homologue Cactus in vivo. CalpA alters the absolute amounts of Cactus protein. Our data indicate, however, that CalpA uses additional mechanisms to regulate NFκB function. We provide evidence that CalpA interacts physically with Cactus, recognizing a Cactus pool that is not bound to Dorsal, a fly NFκB/Rel homologue. We show that proteolytic cleavage by CalpA generates Cactus fragments lacking an N-terminal region required for Toll responsiveness. These fragments are generated in vivo and display properties distinct from those of full-length Cactus. We propose that CalpA targets free Cactus, which is incorporated into and modulates Toll-responsive complexes in the embryo and immune system.
Assuntos
Calpaína/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas I-kappa B/metabolismo , Fosfoproteínas/metabolismo , Proteólise , Receptores Toll-Like/metabolismo , Animais , Padronização Corporal , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/imunologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Corpo Adiposo/citologia , Corpo Adiposo/metabolismo , Sistema Imunitário/metabolismo , Larva/citologia , Larva/metabolismo , Modelos Biológicos , Mutação/genética , Inibidor de NF-kappaB alfa , Fosfoproteínas/química , Ligação ProteicaRESUMO
Sirtuin-1 (SIRT1) is an NAD(+)-dependent protein deacetylase that is sensitive to oxidative signals. Our purpose was to determine whether SIRT1 activity is sensitive to the low molecular weight nitrosothiol, S-nitrosoglutathione (GSNO), which can transduce oxidative signals into physiological responses. SIRT1 formed mixed disulfides with GSNO-Sepharose, and mass spectrometry identified several cysteines that are modified by GSNO, including Cys-67 which was S-glutathiolated. GSNO had no effect on basal SIRT1 deacetylase activity, but inhibited stimulation of activity by resveratrol (RSV) with an IC(50) of 69 microM. These observations indicate that S-glutathiolation of SIRT1 by low concentrations of reactive glutathione can modulate its enzymatic activity.
Assuntos
Glutationa/metabolismo , Glutationa/farmacologia , S-Nitrosoglutationa/metabolismo , Sirtuína 1/metabolismo , Linhagem Celular , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Glutationa/química , Humanos , Compostos Nitrosos/química , Compostos Nitrosos/metabolismo , Compostos Nitrosos/farmacologia , Oxirredução , Proteínas/metabolismo , Resveratrol , Sirtuína 1/química , Estilbenos/farmacologiaRESUMO
The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies for selective enrichment, problems are still encountered due to sample loss or poor selectivity of enrichment. Here we show that the recently introduced fluorous chemistry approach has attractive properties as an alternative method for selective enrichment. Through appending a perfluorine group to the target peptide, it is possible to dramatically increase the peptide's hydrophobicity and thus enable facile separation of labeled from non-labeled peptides. Use of reversed-phase chromatography allowed for improved peptide recovery in comparison with results obtained using the formerly reported fluorous bonded phase methods. Furthermore, this approach also allowed for on-line separation and identification of both labeled and unlabeled peptides in a single experiment. The net result is an increase in the confidence of protein identification by tandem mass spectrometry (MS2) as all peptides and subsequent information are retained. Successful off-line and on-line enrichment of cysteine-containing peptides was obtained, and high quality MS2 spectra were obtained by tandem mass spectrometry due to the stability of the tag, allowing for facile identification via standard database searching. We believe that this strategy holds great promise for selective enrichment and identification of low abundance target proteins or peptides.
Assuntos
Cromatografia de Fase Reversa/métodos , Peptídeos/química , Peptídeos/isolamento & purificação , Animais , Bovinos , Cromatografia de Fase Reversa/instrumentação , Mioglobina/química , Espectrometria de Massas por Ionização por Electrospray , Coloração e RotulagemRESUMO
In addition to haemostasis, platelets mediate inflammation and clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve cytoskeletal rearrangements and release of granular content. Stimulation of the immune Toll-like receptor 2 (TLR2) on the platelet surface, activates phosphoinositide-3-kinase (PI3K) and causes platelet activation and platelet-dependent thrombosis. It remains unknown if platelet activation by immune versus thrombotic pathways leads to the differential regulation of signal transduction, protein-protein interactions, and alpha-granule release, and the physiological relevance of these potential differences. We investigated these processes after immune versus thrombotic platelet stimulation. We examined selected signalling pathways and found that phosphorylation kinetics of Akt, ERK1/2 and p38 differed dramatically between agonists. Next, we investigated platelet protein-protein interactions by mass spectrometry (MS)-based proteomics specifically targeting cytosolic factor XIIIa (FXIIIa) because of its function as a cytoskeleton-crosslinking protein whose binding partners have limited characterisation. Four FXIIIa-binding proteins were identified, two of which are novel interactions: FXIIIa-focal adhesion kinase (FAK) and FXIIIa-gelsolin. The binding of FAK to FXIIIa was found to be altered differentially by immune versus thrombotic stimulation. Lastly, we studied the effect of thrombin versus Pam(3)CSK(4) stimulation on alpha-granule release and observed differential release patterns for selected granule proteins and decreased fibrin clot formation compared with thrombin. The inhibition of PI3K caused a decrease in protein release after Pam(3)CSK(4)- but not after thrombin-stimulation. In summary, stimulation of platelets by either thrombotic or immune receptors leads to markedly different signalling responses and granular protein release consistent with differential contribution to coagulation and thrombosis.
Assuntos
Plaquetas , Ativação Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Trombose , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Plaquetas/citologia , Plaquetas/imunologia , Plaquetas/metabolismo , Comunicação Celular/fisiologia , Grânulos Citoplasmáticos/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator XIIIa/metabolismo , Humanos , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Ativação Plaquetária/efeitos dos fármacos , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trombina/metabolismo , Trombina/farmacologia , Trombose/imunologia , Trombose/metabolismo , Trombose/patologia , Receptor 2 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The extreme pathological diversity of non-Hodgkin's lymphomas has made their accurate histological assessment difficult. New diagnostics and treatment modalities are urgently needed for these lymphomas, particularly in drug development for cancer-specific targets. Previously, we showed that a subset of B cell lymphoma, diffuse large B cell lymphoma, may be characterized by two major, orthogonal axes of gene expression: one set of transcripts that is differentially expressed between resting and proliferating, nonmalignant cells (ie, a "proliferative signature") and another set that is expressed only in proliferating malignant cells (ie, a "cancer signature"). A differential proteomic analysis of B cell proliferative states, similar to previous transcriptional profiling analyses, holds great promise either to reveal novel factors that participate in lymphomagenesis or to define biomarkers of onset or progression. Here, we use a murine model of diffuse large B cell lymphoma to conduct unbiased two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic analyses of malignant proliferating B cells and tissue-matched, normal resting, or normal proliferating cells. We show that the expression patterns of particular proteins or isoforms across these states fall into eight specific trends that provide a framework to identify malignancy-associated biomarkers and potential drug targets, a signature proteome. Our results support the central hypothesis that clusters of proteins of known function represent a panel of expression markers uniquely associated with malignancy and not normal proliferation.