RESUMO
Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.
Assuntos
Autofagia/genética , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , AMP Cíclico/genética , Neoplasias/genética , Animais , Anoikis/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Estresse do Retículo Endoplasmático , Glucose/deficiência , Glucose/metabolismo , Glutamina/metabolismo , Glicólise , Humanos , Camundongos , Neoplasias/metabolismo , Inanição , TranscriptomaRESUMO
Snf1, the yeast AMP-activated kinase homolog, regulates the expression of several genes involved in adaptation to glucose limitation and in response to cellular stresses. We previously demonstrated that Snf1 interacts with Swi6, the regulatory subunit of SBF and MBF complexes, and activates CLB5 transcription. Here we report that, in α-factor synchronized cells in 2% glucose, the loss of the Snf1 catalytic subunit impairs the binding of SBF and MBF complexes and the subsequent recruitment of the FACT complex and RNA Polymerase II to promoters of G1-genes. By using an analog-sensitive allele of SNF1, SNF1(as)(I132G), encoding a protein whose catalytic activity is selectively inhibited in vivo by 2-naphthylmethyl pyrazolopyrimidine 1, we show that the inhibition of Snf1 catalytic activity affects the expression of G1-genes causing a delayed entrance into S phase in cells synchronized in G1 phase by α-factor treatment or by elutriation. Moreover, Snf1 is detected in immune complexes of Rpb1, the large subunit of RNA Polymerase II, and is present at both promoters and coding regions of SBF- and MBF-regulated genes 20min after α-factor release, suggesting a direct role for Snf1 in the activation of the G1-regulon transcription.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Biocatálise/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/genética , Glucose/farmacologia , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transcrição Gênica/efeitos dos fármacosRESUMO
In Saccharomyces cerevisiae, the entrance into S phase requires the activation of a specific burst of transcription, which depends on SBF (SCB binding factor, Swi4/Swi6) and MBF (MCB binding factor, Mbp1/Swi6) complexes. CK2 is a pleiotropic kinase involved in several cellular processes, including the regulation of the cell cycle. CK2 is composed of two catalytic subunits (α and α') and two regulatory subunits (ß and ß'), both of which are required to form the active holoenzyme. Here we investigate the function of the CK2 holoenzyme in Start-specific transcription. The ckb1Δ ckb2Δ mutant strain, bearing deletions of both genes encoding CK2 regulatory subunits, shows a delay of S-phase entrance due to a severe reduction of the expression of SBF- and MBF-dependent genes. This transcriptional defect is caused by an impaired recruitment of Swi6 and Swi4 to G1 gene promoters. Moreover, CK2 α and ß' subunits interact with RNA polymerase II, whose binding to G1 promoters is positively regulated by the CK2 holoenzyme. Collectively, these findings suggest a novel role for the CK2 holoenzyme in the activation of G1 transcription.
Assuntos
Caseína Quinase II/metabolismo , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/metabolismo , Sítio de Iniciação de Transcrição , Caseína Quinase II/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G1 , Deleção de Genes , Holoenzimas/genética , Holoenzimas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/metabolismo , Fase S , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in ß4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 µs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the ß4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.
Assuntos
Proteínas de Saccharomyces cerevisiae/química , Enzimas de Conjugação de Ubiquitina/química , Complexos Ubiquitina-Proteína Ligase/química , Ubiquitina/química , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Humanos , Simulação de Dinâmica Molecular , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fosforilação , Análise de Componente Principal , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
RATIONALE: Quantitative phosphoproteomics represents a front line for functional proteomics and hence for systems biology. Here we present a new application of the surface-activated chemical ionization (SACI) technology for quantitative phosphoproteomics analysis. The main advantages of SACI-MS technology are high sensitivity, quantitative accuracy and matrix effect reduction, which allow quantitative estimations. METHODS: A SACI-MS approach was used to investigate the quantitative in vivo phosphorylation of the cyclin-dependent kinase inhibitor Sic1, a low-abundance protein of Saccharomyces cerevisiae, which is phosphorylated on Ser201 by casein kinase 2 (CK2) and compared its phosphorylation status in cells growing in two different carbon sources (glucose or ethanol). RESULTS: Our relative quantification indicated that the Sic1-Ser201 phosphorylation level is about 2-fold higher in ethanol- than in glucose-growing cells, proportional to the Sic1 protein level. This finding is coherent with results of western blot analysis using anti-phospho-Ser201-specific antibody, validating the results obtained with this new SACI approach. CONCLUSIONS: The findings presented in this paper indicate that the innovative LC/SACI-MS method, coupled with immunoprecipitation, is a powerful device to obtain quantitative information on the phosphorylation state of low abundance proteins.
Assuntos
Proteínas Inibidoras de Quinase Dependente de Ciclina/análise , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Espectrometria de Massas/métodos , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Caseína Quinase II/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/química , Etanol/metabolismo , Fase G1 , Glucose/metabolismo , Imunoprecipitação , Dados de Sequência Molecular , Fosforilação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Serina/análise , Serina/químicaRESUMO
CK2 is a highly conserved protein kinase involved in different cellular processes, which shows a higher activity in actively proliferating mammalian cells and in various types of cancer and cancer cell lines. We recently demonstrated that CK2 activity is strongly influenced by growth rate in yeast cells as well. Here, we extend our previous findings and show that, in cells grown in either glucose or ethanol-supplemented media, CK2 presents no alteration in K(m) for both the ATP and the peptide substrate RRRADDSDDDDD, while a significant increase in V (max) is observed. In chemostat-grown cells, no difference of CK2 activity was observed in cells grown at the same dilution rate in media supplemented with either ethanol or glucose, excluding the contribution of carbon metabolism on CK2 activity. By using the eIF2ß-derived peptide, which can be phosphorylated by the holoenzyme but not by the free catalytic subunits, we show that the holoenzyme activity requires the concurrent presence of both ß and ß' encoding genes. Finally, conditions of nitrogen deprivation leading to a G0-like arrest result in a decrease of total CK2 activity, but have no effect on the activity of the holoenzyme. These findings newly indicate a regulatory role of ß and ß' subunits of CK2 in the nutrient response.
Assuntos
Caseína Quinase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Carbono/farmacologia , Etanol/farmacologia , Glucose/farmacologia , Holoenzimas/metabolismo , Dados de Sequência Molecular , Nitrogênio/deficiência , Nitrogênio/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Identification of interacting proteins in stable complexes is essential to understand the mechanisms that regulate cellular processes at the molecular level. Transcription initiation in prokaryotes requires coordinated protein-protein and protein-DNA interactions that often involve one or more transcription factors in addition to RNA polymerase (RNAP) subunits. The RNAP alpha subunit (RNAPalpha) is a key regulatory element in gene transcription and functions through direct interaction with other proteins to control all stages of this process. A clear description of the RNAPalpha protein partners should greatly increase our understanding of transcription modulation. A functional proteomics approach was employed to investigate protein components that specifically interact with RNAPalpha. A tagged form of Escherichia coli RNAPalpha was used as bait to determine the molecular partners of this subunit in a whole-cell extract. Among other interacting proteins, 50S ribosomal protein L2 (RPL2) was clearly identified by mass spectrometry. The direct interaction between RNAPalpha and RPL2 was confirmed both in vivo and in vitro by performing coimmunoprecipitation and bacterial two-hybrid experiments. The functional role of this interaction was also investigated in the presence of a ribosomal promoter by using a beta-galactosidase gene reporter assay. The results clearly demonstrated that RPL2 was able to increase beta-galactosidase expression only in the presence of a specific ribosomal promoter, whereas it was inactive when it was assayed with an unrelated promoter. Interestingly, other ribosomal proteins (L1, L3, L20, and L27) did not have any effect on rRNA expression. The findings reported here strongly suggest that in addition to its role in ribosome assembly the highly conserved RPL2 protein plays a specific and direct role in regulation of transcription.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas Ribossômicas/metabolismo , Transcrição Gênica , Genes Reporter , Genes de RNAr , Espectrometria de Massas , Regiões Promotoras Genéticas , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
In this note we discuss how, by using budding yeast as model organism (as has been done in the past for biochemical, genetics and genomic studies), the integration of "omics" sciences and more specifically of proteomics with systems biology offers a very profitable approach to elucidating regulatory circuits of complex biological functions.
Assuntos
Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Ciclo Celular , Proteômica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Biologia de SistemasRESUMO
CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in exponential growing batch cultures at faster growth rate, while localization of catalytic and regulatory subunits is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol-grown cells appear to depend on both increase in molecule number and k(cat). Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism.
Assuntos
Caseína Quinase II/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Carbono/metabolismo , Núcleo Celular/enzimologia , Saccharomyces cerevisiae/enzimologiaRESUMO
mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. Although several mechanisms have been identified, emerging evidence suggests that most transcripts reach the protein functional site by moving along cytoskeleton elements. We demonstrated previously that mRNA for mitochondrial ribosomal proteins are asymmetrically distributed in the cytoplasm, and that localization in the proximity of mitochondria is mediated by the 3'-UTR. Here we show by biochemical analysis that these mRNA transcripts are associated with the cytoskeleton through the microtubule network. Cytoskeleton association is functional for their intracellular localization near the mitochondrion, and the 3'-UTR is involved in this cytoskeleton-dependent localization. To identify the minimal elements required for localization, we generated DNA constructs containing, downstream from the GFP gene, deletion mutants of mitochondrial ribosomal protein S12 3'-UTR, and expressed them in HeLa cells. RT-PCR analysis showed that the localization signals responsible for mRNA localization are located in the first 154 nucleotides. RNA pull-down assays, mass spectrometry, and RNP immunoprecipitation assay experiments, demonstrated that mitochondrial ribosomal protein S12 3'-UTR interacts specifically with TRAP1 (tumor necrosis factor receptor-associated protein1), hnRNPM4 (heterogeneous nuclear ribonucleoprotein M4), Hsp70 and Hsp60 (heat shock proteins 70 and 60), and alpha-tubulin in vitro and in vivo.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Regiões 3' não Traduzidas , Chaperonina 60/metabolismo , Citoesqueleto/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Tubulina (Proteína)/metabolismoRESUMO
This chapter focuses on the development of new proteomic approaches based on classical biochemical procedures coupled with new mass spectrometry methods to study the phosphorylation, the most important and abundant PTMs in modulating protein activity and propagating signals within cellular pathways and networks. These phosphoproteome studies aim at comprehensive analysis of protein phosphorylation by identification of the phosphoproteins, exact localization of phosphorylated residues, and preferably quantification of the phosphorylation. Because of low stoichiometry, heterogeneity, and low abundance, enrichment of phosphopeptides is an important step of this analysis. The first section is focused on the development of new enrichment methods coupled to mass spectrometry. Thus, improved approach, based on simple chemical manipulations and mass spectrometric procedures, for the selective analysis of phosphoserine and phosphothreonine in protein mixtures, following conversion of the peptide phosphate moiety into DTT derivatives, is described. However the major aim of this work is devoted to the use of isotopically labelled DTT, thus allowing a simple and direct quantitative MS analysis. The final part of the work is focused on the development of a strategy to study phosphorylation without preliminary enrichment but using the high performance of a novel hybrid mass spectrometer linear ion trap.
Assuntos
Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Fosfoproteínas/análise , Proteoma/análise , Animais , Humanos , Espectrometria de Massas/tendências , Modelos Biológicos , Fosforilação , Proteínas Quinases/metabolismoRESUMO
BACKGROUND: In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic alpha-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular proteolytic activity. RESULTS: P. haloplanktis TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of P. haloplanktis TAC125 in which the gspE gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-beta-lactamase chimera. CONCLUSION: Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic alpha-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the gspE mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium.
RESUMO
Human body fluids have been rediscovered in the post-genomic era as a great source of biological markers and perhaps as source of potential biomarkers of disease. Recently, it has been found that not only proteins but also peptides and their modifications can be indicators of early pathogenic processes. This paper reports the identification of free phosphopeptides in human fluids using an improved IMAC strategy coupled to iterative mass spectrometry-based scanning techniques (neutral loss, precursor ion, multiple reaction monitoring). Many peptides were detected in the enriched extract samples when submitted to the MS-integrated strategy, whereas they were not detected in the initial extract samples. The combination of the IMAC-modified protocol with selective "precursor ion" and constant "neutral loss" triple quadrupole scan modes confers a high sensitivity on the analysis, allowing rapid phosphopeptide identification and characterization, even at low concentrations. To the best of our knowledge this work represents the first report exclusively focused on the detection of free phosphorylated peptides in biological fluids.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fosfopeptídeos/análise , Saliva/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Caseínas/análise , Caseínas/química , Precipitação Química , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/química , Fibrinogênio/análise , Fibrinogênio/química , Humanos , Nanotecnologia/métodos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fosfopeptídeos/sangue , Fosfopeptídeos/metabolismo , Fosfopeptídeos/urina , Saliva/metabolismo , Análise de Sequência de Proteína/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
SIGNIFICANCE: Histone deacetylases (HDACs) activity and cell metabolism are considered important targets for cancer therapy, as both are deregulated and associated with the onset and maintenance of tumors. RECENT ADVANCES: Besides the classical function of HDACs as HDAC enzymes controlling the transcription, it is becoming increasingly evident that these proteins are involved in the regulation of several other cellular processes by their ability to deacetylate hundreds of proteins with different functions in both the cytoplasm and the nucleus. Importantly, recent high-throughput studies have identified as important target proteins several enzymes involved in different metabolic pathways. Conversely, it has been also shown that metabolic intermediates may control HDACs activity. Consequently, the acetylation/deacetylation of metabolic enzymes and the ability of metabolic intermediates to modulate HDACs may represent a cross-talk connecting cell metabolism, transcription, and other HDACs-controlled processes in physiological and pathological conditions. CRITICAL ISSUES: Since metabolic alterations and HDACs deregulation are important cancer hallmarks, disclosing connections among them may improve our understanding on cancer mechanisms and reveal novel therapeutic protocols against this disease. FUTURE DIRECTIONS: High-throughput metabolic studies performed by using more sophisticated technologies applied to the available models of conditional deletion of HDACs in cell lines or in mice will fill the gap in the current understanding and open directions for future research.
Assuntos
Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Redes e Vias Metabólicas , Neoplasias/metabolismo , Acetilação , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Neoplasias/tratamento farmacológicoRESUMO
Phosphoproteomics, nowadays, represents a front line in functional proteomics as testified by the number of papers recently appearing in the literature. In an attempt to improve and simplify the methods so far suggested we have set up a simple isotope-coded approach to label and quantitate phospho-Ser/-Thr residues in protein mixtures. First of all, after appropriate oxidation of cysteine/cystine residues followed by tryptic hydrolysis, we have optimised and simplified the beta-elimination reaction to get the corresponding alkene moiety from the phosphate esters. This was achieved by (a) separating the elimination reaction from the addition reaction, (b) the use of Ba(OH)(2) as alkali reagent and (c) its further elimination by the simple addition of solid CO(2) to the peptide mixture. The Michael reaction was then performed, after the removal of BaCO(3) by centrifugation, by adding dithiothreitol (DTT) to the peptide mixture. Finally, the direct purification of the modified phosphopeptides was performed on a thiol-sepharose column. The availability of fully deuterated DTT, introducing a 6 Da difference with respect to the non-deuterated species, allows quantitation of the differential extent of signalling modification when analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and liquid chromatography/mass spectrometry. The entire procedure has been set up by using bovine alpha-casein, and resulted in the identification of all the phosphorylated tryptic peptides, including the tetraphosphorylated peptides, which escaped all previously reported procedures
Assuntos
Ditiotreitol/química , Espectrometria de Massas/métodos , Fosfoproteínas/química , Sequência de Aminoácidos , Animais , Caseínas/química , Caseínas/metabolismo , Bovinos , Marcação por Isótopo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Compostos de Sulfidrila/química , Tripsina/metabolismoRESUMO
Cancer cells generally rely mostly on glycolysis rather than oxidative phosphorylation (OXPHOS) for ATP production. In fact, they are particularly sensitive to glycolysis inhibition and glucose depletion. On the other hand mitochondrial dysfunctions, involved in the onset of the Warburg effect, are sometimes also associated with the resistance to apoptosis that characterizes cancer cells. Therefore, combined treatments targeting both glycolysis and mitochondria function, exploiting peculiar tumor features, might be lethal for cancer cells. In this study, we show that glucose deprivation and mitochondrial Complex I inhibitors synergize in inducing cancer cell death. In particular, our results reveal that low doses of Complex I inhibitors, ineffective on immortalized cells and in high glucose growth, become specifically cytotoxic on cancer cells deprived of glucose. Importantly, the cytotoxic effect of the inhibitors on cancer cells is strongly enhanced by forskolin, a PKA pathway activator, that we have previously shown to stimulate OXPHOS. Taken together, we demonstrate that induction in cancer cells of a switch from a glycolytic to a more respirative metabolism, obtained by glucose depletion or mitochondrial activity stimulation, strongly increases their sensitivity to low doses of mitochondrial Complex I inhibitors. Our findings might be a valuable approach to eradicate cancer cells.
RESUMO
It has been recently hypothesized that BAG3 protein, a co-chaperone of Hsp70/Hsc70, is involved in the regulation of several cell processes, such as apoptosis, autophagy and cell motility. Following the identification of Hsc70/Hsp70, further BAG3 molecular partners such as PLC-gamma and HspB8 were likewise identified, thus contributing to the characterization of the mechanisms and the biological roles carried out by this versatile protein. By using a His-tagged BAG3 protein as bait, we fished out and identified the cytosolic chaperonin CCT, a new unreported BAG3 partner. The interaction between BAG3 and CCT was confirmed and characterized by co-immunoprecipitation experiments and surface plasmon resonance techniques. Furthermore, our analyses showed a slower CCT association and a faster dissociation with a truncated form of BAG3 containing the BAG domain, thus indicating that other protein regions are essential for a high-affinity interaction. ATP or ADP does not seem to significantly influence the chaperonin binding to BAG3 protein. On the other hand, our experiments showed that BAG3 silencing by small interfering RNA slowed down cell migration and influence the availability of correctly folded monomeric actin, analyzed by DNAse I binding assays and latrunculin A depolymerization studies. To our knowledge, this is the first report showing a biologically relevant interaction between the chaperonin CCT and BAG3 protein, thus suggesting interesting involvement in the folding processes regulated by CCT.
Assuntos
Actinas/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Chaperonina com TCP-1/metabolismo , Dobramento de Proteína , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Movimento Celular , Citoesqueleto/efeitos dos fármacos , Desoxirribonuclease I/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imunoprecipitação , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , RNA Interferente Pequeno , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em TandemRESUMO
The ubiquitin-conjugating enzyme Cdc34 was recently shown to be phosphorylated by CK2 on the C-terminal tail. Here we present novel findings indicating that in budding yeast CK2 phosphorylates Cdc34 within the N-terminal catalytic domain. Specifically, we show, by direct mass spectrometry analysis, that Cdc34 is phosphorylated in vitro and in vivo by CK2 on Ser130 and Ser167, and that the phosphoserines 130 and 167 are not present after CK2 inactivation in a cka1Deltacka2-8(ts) strain. CK2 phosphorylation of Ser130 and Ser167 strongly stimulates Cdc34 ubiquitin charging in vitro. The Cdc34(S130AS167A) mutant shows a basal ubiquitin charging activity which is indistinguishable from that of wild type but is not activated by CK2 phosphorylation and its expression fails to complement a cdc34-2(ts) yeast strain, supporting a model in which activation of Cdc34 involves CK2-mediated phosphorylation of its catalytic domain.
Assuntos
Fase S/fisiologia , Saccharomyces cerevisiae/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Citometria de Fluxo , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Alinhamento de Sequência , Serina/metabolismo , Enzimas de Conjugação de Ubiquitina , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
Characterization of the membrane proteome is particularly intriguing since a better knowledge in this field might lead to new insights into the function of different membrane systems. Despite the biological relevance of surface proteins however, their characterization still remains a challenging task. Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Hence, surface proteins of Gram-negative bacteria contain proteins that might be good targets for drugs, antimicrobials or detection systems and they may become components of effective vaccines. In this respect, Escherichia coli has been chosen as a model organism for several structural and functional studies aimed at understanding the biophysical and biochemical organization of proteins in Gram-negative cell walls. Here we present first results for the identification of bacterial surface exposed proteins in E. coli K12 based on the use of dansyl chloride labelling coupled with bidimensional tandem mass spectrometry exploiting the advantage of precursor ion/MS3 scan modes. This procedure resulted in a promising, simple, and rapid strategy for the identification of membrane proteins in E. coli as model organism, thus avoiding time-consuming procedures based on two-dimensional liquid chromatography and electrophoresis. The proteins identified could be grouped into five major families: outer membrane (29 proteins), lipoproteins (6 proteins), transmembrane (43 proteins) families.
Assuntos
Cromatografia Líquida/métodos , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Mapeamento de Peptídeos/métodos , Proteoma/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Protein phosphorylation regulates many cellular processes and pathways, such as cell cycle progression, signal transduction cascades and gene expression. Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel approach to label selectively phospho-Ser/-Thr residues by exploiting the features of a novel linear ion trap mass spectrometer. Using dansyl labelling and MS3 fragmentation, we developed a method useful for the large-scale proteomic profiling of phosphorylation sites. The new residues in the sequence were stable and easily identifiable under general conditions for tandem mass spectrometric sequencing.