RESUMO
Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO(3)(-). The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into three-dimensional collagen matrix.
Assuntos
Melanoma/metabolismo , Proteômica , Proteínas Proto-Oncogênicas B-raf/metabolismo , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Espectrometria de Massas , Mutação , Fosfoproteínas/análise , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/fisiologia , Especificidade por SubstratoRESUMO
We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability, probed with circular dichroism, established an accessible temperature range of 10 degrees C to 55 degrees C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, DeltaH degrees (unf(avg)). Most regions in the enzyme show DeltaH degrees (unf(avg)) < or = 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for DeltaH degrees (unf(avg)) are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate.
Assuntos
Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deutério , Estabilidade Enzimática , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Hidrogênio , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Movimento (Física) , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Tetra-Hidrofolato Desidrogenase/genética , TermodinâmicaRESUMO
The technique of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog, Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen-deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 °C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 10074].
Assuntos
Tetra-Hidrofolato Desidrogenase/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Medição da Troca de Deutério , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Homologia Estrutural de Proteína , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , TermodinâmicaRESUMO
When analyzing proteins in complex samples using tandem mass spectrometry of peptides generated by proteolysis, the inference of proteins can be ambiguous, even with well-validated peptides. Unresolved questions include whether to show all possible proteins vs a minimal list, what to do when proteins are inferred ambiguously, and how to quantify peptides that bridge multiple proteins, each with distinguishing evidence. Here we describe IsoformResolver, a peptide-centric protein inference algorithm that clusters proteins in two ways, one based on peptides experimentally identified from MS/MS spectra, and the other based on peptides derived from an in silico digest of the protein database. MS/MS-derived protein groups report minimal list proteins in the context of all possible proteins, without redundantly listing peptides. In silico-derived protein groups pull together functionally related proteins, providing stable identifiers. The peptide-centric grouping strategy used by IsoformResolver allows proteins to be displayed together when they share peptides in common, providing a comprehensive yet concise way to organize protein profiles. It also summarizes information on spectral counts and is especially useful for comparing results from multiple LC-MS/MS experiments. Finally, we examine the relatedness of proteins within IsoformResolver groups and compare its performance to other protein inference software.
Assuntos
Mineração de Dados/métodos , Fragmentos de Peptídeos/análise , Isoformas de Proteínas , Proteômica/métodos , Algoritmos , Cromatografia Líquida , Bases de Dados de Proteínas , Humanos , Fragmentos de Peptídeos/química , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Software , Espectrometria de Massas em Tandem , Tripsina/metabolismoRESUMO
Identifying peptides from mass spectrometric fragmentation data (MS/MS spectra) using search strategies that map protein sequences to spectra is computationally expensive. An alternative strategy uses direct spectrum-to-spectrum matching against a reference library of previously observed MS/MS that has the advantage of evaluating matches using fragment ion intensities and other ion types than the simple set normally used. However, this approach is limited by the small sizes of the available peptide MS/MS libraries and the inability to evaluate the rate of false assignments. In this study, we observed good performance of simulated spectra generated by the kinetic model implemented in MassAnalyzer (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922; Zhang, Z. (2005) Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges. Anal. Chem. 77, 6364-6373) as a substitute for the reference libraries used by the spectrum-to-spectrum search programs X!Hunter and BiblioSpec and similar results in comparison with the spectrum-to-sequence program Mascot. We also demonstrate the use of simulated spectra for searching against decoy sequences to estimate false discovery rates. Although we found lower score discrimination with spectrum-to-spectrum searches than with Mascot, particularly for higher charge forms, comparable peptide assignments with low false discovery rate were achieved by examining consensus between X!Hunter and Mascot, filtering results by mass accuracy, and ignoring score thresholds. Protein identification results are comparable to those achieved when evaluating consensus between Sequest and Mascot. Run times with large scale data sets using X!Hunter with the simulated spectral library are 7 times faster than Mascot and 80 times faster than Sequest with the human International Protein Index (IPI) database. We conclude that simulated spectral libraries greatly expand the search space available for spectrum-to-spectrum searching while enabling principled analyses and that the approach can be used in consensus strategies for large scale studies while reducing search times.
Assuntos
Simulação por Computador , Espectrometria de Massas , Biblioteca de Peptídeos , Proteínas/análise , Sequência de Aminoácidos , Linhagem Celular Tumoral , Bases de Dados de Proteínas , Humanos , Peptídeos/química , Proteínas/química , Curva ROC , Padrões de Referência , SoftwareRESUMO
RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named "mediator of RhoA-dependent invasion (MRDI)," that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Melanoma , Metionina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Humanos , Melanoma/enzimologia , Melanoma/patologia , Metionina/química , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Estrutura Molecular , Invasividade Neoplásica , Metástase Neoplásica , Proteômica/métodos , Interferência de RNA , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Transdução de Sinais/fisiologia , Transplante Heterólogo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Duplication of the Saccharomyces cerevisiae spindle pole body (SPB) once per cell cycle is essential for bipolar spindle formation and accurate chromosome segregation during mitosis. We have investigated the role that the major yeast cyclin-dependent kinase Cdc28/Cdk1 plays in assembly of a core SPB component, Spc42, to better understand how SPB duplication is coordinated with cell cycle progression. Cdc28 is required for SPB duplication and Spc42 assembly, and we found that Cdc28 directly phosphorylates Spc42 to promote its assembly into the SPB. The Mps1 kinase, previously shown to regulate Spc42 phosphorylation and assembly, is also a Cdc28 substrate, and Cdc28 phosphorylation of Mps1 is needed to maintain wild-type levels of Mps1 in cells. Analysis of nonphosphorylatable mutants in SPC42 and MPS1 indicates that direct Spc42 phosphorylation and indirect regulation of Spc42 through Mps1 are two overlapping pathways by which Cdc28 regulates Spc42 assembly and SPB duplication during the cell cycle.
Assuntos
Proteína Quinase CDC2/fisiologia , Proteína Quinase CDC28 de Saccharomyces cerevisiae/fisiologia , Proteínas do Citoesqueleto/fisiologia , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Fuso Acromático , Alelos , Ciclo Celular , Centrossomo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Espectrometria de Massas , Mitose , Modelos Biológicos , Mutação , Fosfoproteínas/metabolismo , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/química , Temperatura , Treonina/químicaRESUMO
Urothelial plaques consist of four major uroplakins (Ia, Ib, II, and III) that form two-dimensional crystals covering the apical surface of urothelium, and provide unique opportunities for studying membrane protein assembly. Here, we describe a novel 35-kD urothelial plaque-associated glycoprotein that is closely related to uroplakin III: they have a similar overall type 1 transmembrane topology; their amino acid sequences are 34% identical; they share an extracellular juxtamembrane stretch of 19 amino acids; their exit from the ER requires their forming a heterodimer with uroplakin Ib, but not with any other uroplakins; and UPIII-knockout leads to p35 up-regulation, possibly as a compensatory mechanism. Interestingly, p35 contains a stretch of 80 amino acid residues homologous to a hypothetical human DNA mismatch repair enzyme-related protein. Human p35 gene is mapped to chromosome 7q11.23 near the telomeric duplicated region of Williams-Beuren syndrome, a developmental disorder affecting multiple organs including the urinary tract. These results indicate that p35 (uroplakin IIIb) is a urothelial differentiation product structurally and functionally related to uroplakin III, and that p35-UPIb interaction in the ER is an important early step in urothelial plaque assembly.
Assuntos
Glicoproteínas de Membrana/metabolismo , Urotélio/crescimento & desenvolvimento , Urotélio/metabolismo , Sequência de Aminoácidos , Animais , Biomarcadores , Bovinos , Células Cultivadas , Cromossomos Humanos Par 7 , Dimerização , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Alinhamento de Sequência , Tetraspaninas , Distribuição Tecidual , Uroplaquina III , Uroplaquina Ib , Urotélio/citologiaRESUMO
The allosteric regulation of protein kinases serves as an efficient strategy for molecular communication, event coupling and interconversion between catalytic states. Recent co-crystal structures have revealed novel ways in which kinases control activity and substrate specificity following phosphorylation, dimerization, or binding to regulatory proteins, substrates and scaffolds. In addition, hydrogen exchange coupled with mass spectrometry is emerging as a complementary strategy to probe the solution behavior of kinases; recent results have shown that allosteric regulation may involve transitions in protein motions as well as structural rearrangements.
Assuntos
Proteínas Quinases/química , Proteínas Quinases/metabolismo , Regulação Alostérica , Sítio Alostérico , Deutério , Dimerização , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hidrogênio , Espectrometria de Massas , Modelos Biológicos , Modelos Moleculares , Estrutura Quaternária de Proteína , Transdução de SinaisRESUMO
The information from genome sequencing provides a new framework for a systems-wide understanding of protein networks and cellular function. Whereas microarray technologies provide information about global gene expression within cells, complementary proteomic strategies monitor expression of proteins and their posttranslational modifications. Improved technologies that have emerged for comprehensive and high-throughput protein analysis yield novel insights into cell regulation.
Assuntos
Proteômica/métodos , Transdução de Sinais , Animais , Humanos , Análise Serial de Proteínas , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Structural and kinetic studies have provided extensive information about the molecular mechanisms of kinase activation by phosphorylation. However, it is still unclear how changes in protein dynamics and flexibility contribute to catalytic function. Mass spectrometry was used to probe changes in hydrogen/deuterium exchange in the MAP kinase, ERK2, in the presence and absence of the ATP analogue, AMP-PNP. In both active and inactive forms of ERK2, protection from hydrogen exchange by AMP-PNP binding was observed within conserved ATP binding motifs in the N-terminal lobe, which are known to directly interact with nucleotide in various protein kinases. In contrast, higher protection from exchange by AMP-PNP was observed in active ERK2 compared to inactive ERK2, in a region corresponding to the conserved DFG motif, which is located in the C-terminal lobe and coordinates Mg2+ at the catalytic site. Thus, AMP-PNP binding simultaneously protects residues within the N and C terminus in the active form of ERK2, but not the inactive form. This demonstrates that ERK2 binds nucleotide in two modes, in which active ERK2 adopts a closed conformation following nucleotide binding in solution, while inactive ERK2 adopts an open conformation. The finding provides novel evidence that phosphorylation of ERK2 facilitates interdomain closure, allowing proper orientation between ATP and substrate to facilitate phosphoryl transfer.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteína Quinase 1 Ativada por Mitógeno/química , Sequência de Aminoácidos , Sítios de Ligação , Ativação Enzimática , Hidrogênio , Cinética , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , SolventesRESUMO
Functional proteomics provides a powerful approach to screen for alterations in protein expression and posttranslational modifications under conditions of human disease. In this study, we use protein screening to examine markers of melanoma progression, by profiling melanocyte versus melanoma cell lines using two-dimensional electrophoresis and mass spectrometry. Eight candidate markers were identified as differentially regulated in transformed cells. In particular, hepatoma-derived growth factor (HDGF) and nucleophosmin B23 were strongly correlated with melanoma. Nucleophosmin B23 is a nucleolar and centrosome-associated protein, which has been implicated as a target for cyclin E/cyclin-dependent kinase 2 (CDK2) in modulating centrosome duplication and cell cycle control. Western blotting of one-dimensional and two-dimensional gels showed that the form of nucleophosmin B23 that is up-regulated in melanoma represents a posttranslationally modified form, most likely reflecting enhanced phosphorylation in the tumor-derived cells. In contrast, Western analysis of HDGF demonstrated increased expression of all forms in melanoma cell lines compared with melanocytes. Immunohistochemical analysis of human tissue biopsies showed strong expression of HDGF in early and late stage melanomas and low expression in melanocytes and nontumorigenic nevi. Interestingly, biopsies of nevi showed a graded effect in which HDGF immunoreactivity was reduced in nevoid nests penetrating deep into the dermis compared with nests at the epidermal-dermal junction, suggesting that HDGF expression in nevi is dependent on epidermal cell interactions. In contrast, biopsies of melanoma showed strong expression of HDGF throughout the tumor, including cells located deeply within dermis. Thus, expression of this antigen likely reports a reduced dependence of protein expression on epidermal interactions.
Assuntos
Melanoma/metabolismo , Melanoma/patologia , Proteômica/métodos , Biomarcadores Tumorais/metabolismo , Catepsina D/biossíntese , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Espectrometria de Massas , Melanócitos/metabolismo , Proteínas Nucleares/metabolismo , Nucleofosmina , Processamento de Proteína Pós-TraducionalRESUMO
The information from genome sequencing provides new approaches for systems-wide understanding of protein networks and cellular function. DNA microarray technologies have advanced to the point where nearly complete monitoring of gene expression is feasible in several organisms. An equally important goal is to comprehensive survey cellular proteomes and profile protein changes under different cellular states. This presents a complex analytical problem, due to the chemical variability between proteins and peptides. Here, we discuss strategies to improve accuracy and sensitivity of peptide identification, distinguish represented protein isoforms, and quantify relative changes in protein abundance.
Assuntos
Biologia Computacional/métodos , Proteínas/análise , Proteômica/métodos , Bases de Dados de Proteínas , Mapeamento de Peptídeos/métodos , Análise Serial de Proteínas , Mapeamento de Interação de Proteínas/métodosRESUMO
Two-dimensional electrophoresis (2-DE) is a powerful technique to differentially display patterns of protein expression and posttranslational modifications, providing a good strategy to monitor molecular responses induced by the activation or inactivation of specific signaling pathways. In this chapter, optimized protocols for 2-DE using extracts from tissue culture are provided. Protocols for in-gel digestion of gel-resolved proteins, which allow protein identification by mass spectrometry are also discussed.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas/análise , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Focalização Isoelétrica , Espectrometria de Massas , Proteínas/química , Proteínas/metabolismo , Coloração pela Prata/métodosRESUMO
BACKGROUND: In normal mammalian epidermis, cell division occurs primarily in the basal layer where cells are attached to the basement membrane. Upon release from the basement membrane, these basal cells stop dividing and begin to differentiate and stratify producing cornified cells expressing differentiation markers, including the keratin bundling protein filaggrin, and cornified envelope proteins. Little is understood about the regulatory mechanisms of these processes. A rat epidermal keratinocyte cell line synthesizing and processing profilaggrin at confluence in a synchronous manner for 4-5 days provides a useful culture model for epidermal differentiation. Profilaggrin expression in this cell line however decreases with passaging, and its processing involves extensive nonspecific proteolysis. OBJECTIVE: Our objective was to identify culture conditions that effect the decrease in profilaggrin expression with passaging and nonspecific proteolysis of profilaggrin in order to study epidermal differentiation more closely. METHOD: The large amount of nonspecific proteolysis suggested autophagocytosis. To test this, cells were cultured in the presence of 3-methyladenine (3-MA). Two known gradients in epidermis are decreasing serum components and increasing calcium concentrations in the upper cell layers. To determine whether these gradients effected processing, cells were cultured in serum/DMEM or in serum-free KGM and under varying external calcium concentrations. Cells were also cultured in presence of aminoguanidine in an attempt to maintain profilaggrin expression with passaging. RESULTS: Profilaggrin expression was enhanced in the presence of 3-MA, with optimum around 6mM. In the absence of aminoguanidine, profilaggrin expression decreased as a function of increasing passage number; in its presence, profilaggrin expression remained high in some, but not in all of the independently maintained cell lines. Thus, culturing in aminoguanidine was necessary, but not sufficient, for sustained ability to express profilaggrin at confluence. Production of filaggrin from profilaggrin was maximized in a serum-free medium with [Ca(2+)] at 5mM. Filaggrin associates with phospholipid vesicles in vitro forming aggregates similar to those seen in vivo, suggesting that filaggrin release induces vesicular aggregation and autophagocytosis. CONCLUSION: We have used a keratinocyte cell line that synthesizes and processes profilaggrin after confluence as a culture model to study epidermal differentiation. In this system profilaggrin processing must be preceded by inhibition of autophagosome formation and/or modulation of vesicular trafficking, and these processes are regulated by epidermal calcium and serum factor gradients.
Assuntos
Cálcio/metabolismo , Técnicas de Cultura/métodos , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Proteínas Filagrinas , Guanidinas/farmacologia , Proteínas de Filamentos Intermediários/efeitos dos fármacos , RatosAssuntos
Eletroforese em Gel Bidimensional/métodos , Proteômica/métodos , Alquilação , Soluções Tampão , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Focalização Isoelétrica/métodos , Espectrometria de Massas/métodos , Oxirredução , Mapeamento de Peptídeos/métodos , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Coloração e Rotulagem/métodos , TripsinaAssuntos
Deutério , Espectrometria de Massas/métodos , Proteínas/química , Cromatografia Líquida de Alta Pressão/métodos , Interpretação Estatística de Dados , Hidrogênio/química , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/química , Modelos Químicos , SoftwareRESUMO
Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38alpha MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38alpha are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38alpha alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38alpha and X-ray structures of phosphorylated p38gamma. Conformational differences account for altered HX within the activation lip, the P+1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (alphaF-alphaG, beta7-beta8), the C-terminal core (alphaE), and the N-terminal core region (beta4-beta5, alphaL16, alphaC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38alpha. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38alpha because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38alpha are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38alpha and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.