RESUMO
Neurofilament light (NfL) concentration in cerebrospinal fluid (CSF) and blood serves as an important biomarker in neurology drug development. Changes in NfL are generally assumed to reflect changes in neuronal damage, while little is known about the clearance of NfL from biofluids. We observed an NfL increase of 3.5-fold in plasma and 5.7-fold in CSF in an asymptomatic individual at risk for genetic prion disease following 6 weeks' treatment with oral minocycline for a dermatologic indication. Other biomarkers remained normal, and proteomic analysis of CSF revealed that the spike was exquisitely specific to neurofilaments. NfL dropped nearly to normal levels 5 weeks after minocycline cessation, and the individual remained free of disease 2 years later. Plasma NfL in dermatology patients was not elevated above normal controls. Dramatically high plasma NfL (>500 pg/mL) was variably observed in some hospitalized individuals receiving minocycline. In mice, treatment with minocycline resulted in variable increases of 1.3- to 4.0-fold in plasma NfL, with complete washout 2 weeks after cessation. In neuron-microglia co-cultures, minocycline increased NfL concentration in conditioned media by 3.0-fold without any visually obvious impact on neuronal health. We hypothesize that minocycline does not cause or exacerbate neuronal damage, but instead impacts the clearance of NfL from biofluids, a potential confounder for interpretation of this biomarker.
RESUMO
Wnt5a-Ror signaling is a conserved pathway that regulates morphogenetic processes during vertebrate development [R. T. Moon et al, Development 119, 97-111 (1993); I. Oishi et al, Genes Cells 8, 645-654 (2003)], but its downstream signaling events remain poorly understood. Through a large-scale proteomic screen in mouse embryonic fibroblasts, we identified the E3 ubiquitin ligase Pdzrn3 as a regulatory target of the Wnt5a-Ror pathway. Upon pathway activation, Pdzrn3 is degraded in a ß-catenin-independent, ubiquitin-proteasome system-dependent manner. We developed a flow cytometry-based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 stability. Epistatically, Pdzrn3 is regulated independently of Kif26b, another Wnt5a-Ror effector. Wnt5a-dependent degradation of Pdzrn3 requires phosphorylation of three conserved amino acids within its C-terminal LNX3H domain [M. Flynn, O. Saha, P. Young, BMC Evol. Biol. 11, 235 (2011)], which acts as a bona fide Wnt5a-responsive element. Importantly, this phospho-dependent degradation is essential for Wnt5a-Ror modulation of cell migration. Collectively, this work establishes a Wnt5a-Ror cell morphogenetic cascade involving Pdzrn3 phosphorylation and degradation.
Assuntos
Proteômica , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , Proteína Wnt-5a/metabolismo , Animais , Movimento Celular , Camundongos , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Proteólise , Reprodutibilidade dos Testes , Ubiquitina/metabolismoRESUMO
Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.
Assuntos
Proteínas Relacionadas à Autofagia/deficiência , Autofagia , Disenteria Bacilar/microbiologia , Imunidade Inata , Macrófagos/microbiologia , Estresse Oxidativo , Proteoma , Proteômica , Shigella flexneri/patogenicidade , Animais , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Modelos Animais de Doenças , Disenteria Bacilar/imunologia , Disenteria Bacilar/metabolismo , Interações Hospedeiro-Patógeno , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Viabilidade Microbiana , Shigella flexneri/imunologia , Shigella flexneri/metabolismo , VirulênciaRESUMO
Targeted protein quantification using liquid chromatography-tandem mass spectrometry with stable isotope-labeled standards is recognized as the gold standard of practice for protein quantification. Such assays, however, can only cover a limited number of proteins, and developing targeted methods for larger numbers of proteins requires substantial investment. Alternatively, large-scale global proteomic experiments along with computational methods such as the "total protein approach" (TPA) have the potential to provide extensive protein quantification. In this study, we compared the TPA-based quantitation of seven major hepatic uptake transporters in four human liver tissue samples using global proteomic data obtained from two multiplexed tandem mass tag experiments (performed in two independent laboratories) to the quantitative data from targeted proteomic assays. The TPA-based quantitation of these hepatic transporters [sodium-taurocholate cotransporting polypeptide (NTCP/SLC10A1), organic anion transporter 2 (OAT2/SLC22A7), OAT7/SLC22A9, organic anion-transporting polypeptide 1B1 (OATP1B1/SLCO1B1), OATP1B3/SLCO1B3, OATP2B1/SLCO2B1, and organic cation transporter (OCT1/SLC22A1)] showed good-to-excellent correlations (Pearson r = 0.74-1.00) to the targeted data. In addition, the values were similar to those measured by targeted proteomics with 71% and 86% of the data sets falling within 3-fold of the targeted data. A comparison of the TPA-based quantifications of enzyme abundances to available literature data showed that the majority of the enzyme quantifications fell within the reference data intervals. In conclusion, these results demonstrate the capability of multiplexed global proteomic experiments to detect differences in protein expression between samples and provide reasonable estimations of protein expression levels.
Assuntos
Transporte Biológico/fisiologia , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/metabolismo , Cromatografia Líquida/métodos , Hepatócitos/metabolismo , Humanos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Obesity is associated with elevated intracellular nitric oxide (NO) production, which promotes nitrosative stress in metabolic tissues such as liver and skeletal muscle, contributing to insulin resistance. The onset of obesity-associated insulin resistance is due, in part, to the compromise of hepatic autophagy, a process that leads to lysosomal degradation of cellular components. However, it is not known how NO bioactivity might impact autophagy in obesity. Here, we establish that S-nitrosoglutathione reductase (GSNOR), a major protein denitrosylase, provides a key regulatory link between inflammation and autophagy, which is disrupted in obesity and diabetes. We demonstrate that obesity promotes S-nitrosylation of lysosomal proteins in the liver, thereby impairing lysosomal enzyme activities. Moreover, in mice and humans, obesity and diabetes are accompanied by decreases in GSNOR activity, engendering nitrosative stress. In mice with a GSNOR deletion, diet-induced obesity increases lysosomal nitrosative stress and impairs autophagy in the liver, leading to hepatic insulin resistance. Conversely, liver-specific overexpression of GSNOR in obese mice markedly enhances lysosomal function and autophagy and, remarkably, improves insulin action and glucose homeostasis. Furthermore, overexpression of S-nitrosylation-resistant variants of lysosomal enzymes enhances autophagy, and pharmacologically and genetically enhancing autophagy improves hepatic insulin sensitivity in GSNOR-deficient hepatocytes. Taken together, our data indicate that obesity-induced protein S-nitrosylation is a key mechanism compromising the hepatic autophagy, contributing to hepatic insulin resistance.
Assuntos
Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/metabolismo , Autofagia , Diabetes Mellitus/metabolismo , Hepatócitos/metabolismo , Resistência à Insulina , Obesidade/fisiopatologia , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Aldeído Oxirredutases/química , Aldeído Oxirredutases/genética , Animais , Células Cultivadas , Cisteína/metabolismo , Diabetes Mellitus/enzimologia , Diabetes Mellitus/patologia , Dieta Hiperlipídica/efeitos adversos , Regulação Enzimológica da Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Lisossomos/enzimologia , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Estresse Nitrosativo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de FusãoRESUMO
Fertilization releases the meiotic arrest and initiates the events that prepare the egg for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phosphoregulation is still unknown. We examined absolute protein and phosphosite dynamics of the fertilization response by mass spectrometry-based proteomics in electroactivated eggs. To do this, we developed an approach for calculating the stoichiometry of phosphosites from multiplexed proteomics that is compatible with dynamic, stable, and multisite phosphorylation. Overall, the data suggest that degradation is limited to a few low-abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium-activated kinases. We identify putative degradation targets and components of the slow block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.
Assuntos
Fertilização/fisiologia , Meiose/fisiologia , Óvulo/fisiologia , Proteoma/metabolismo , Xenopus laevis/fisiologia , Animais , Cálcio/metabolismo , Feminino , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteólise , Xenopus laevis/embriologiaRESUMO
Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/ß-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.
Assuntos
Cinesinas/metabolismo , Transdução de Sinais , Proteína Wnt-5a/metabolismo , Animais , Linhagem Celular , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Cinesinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese/efeitos dos fármacos , Proteômica , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Via de Sinalização Wnt , Proteína Wnt-5a/farmacologia , beta Catenina/metabolismoRESUMO
Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as â¼44% of the mis-spliced cassette exons in C9ORF72 patient brains.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/metabolismo , Dipeptídeos/farmacologia , Demência Frontotemporal/genética , RNA Nuclear Pequeno/metabolismo , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/genética , Expansão das Repetições de DNA , Dipeptídeos/metabolismo , Demência Frontotemporal/imunologia , Demência Frontotemporal/metabolismo , Humanos , Proteômica/métodos , Splicing de RNA , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismoRESUMO
Microsomal cytochromes P450 (P450) require two electrons and two protons for the oxidation of substrates. Although the two electrons can be provided by cytochrome P450 reductase, the second electron can also be donated by cytochrome b5 (b5). The steady-state activity of P450 2B4 is increased up to 10-fold by b5. To improve our understanding of the molecular basis of the stimulatory effect of b5 and to test the hypothesis that b5 stimulates catalysis by more rapid protonation of the anionic ferric hydroperoxo heme intermediate of P450 (Fe3+OOH)- and subsequent formation of the active oxidizing species (Fe+4âO PORâ¢+), we have freeze-quenched the reaction mixture during a single turnover following reduction of oxyferrous P450 2B4 by each of its redox partners, b5 and P450 reductase. The electron paramagnetic resonance spectra of the freeze-quenched reaction mixtures lacked evidence of a hydroperoxo intermediate when b5 was the reductant presumably because hydroperoxo protonation and catalysis occurred within the dead time of the instrument. However, when P450 reductase was the reductant, a hydroperoxo P450 intermediate was observed. The effect of b5 on the enzymatic efficiency in D2O and the kinetic solvent isotope effect under steady-state conditions are both consistent with the ability of b5 to promote rapid protonation of the hydroperoxo species and more efficient catalysis. In summary, by binding to the proximal surface of P450, b5 stimulates the activity of P450 2B4 by enhancing the rate of protonation of the hydroperoxo intermediate and formation of Compound I, the active oxidizing species, which allows less time for side product formation.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromos b5/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Prótons , Animais , Biocatálise , Família 2 do Citocromo P450/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Elétrons , Hidrogenação , Cinética , Modelos Biológicos , NAD/metabolismo , Oxirredução , Ligação Proteica , Coelhos , Especificidade por SubstratoRESUMO
The existence of the ocular microbiota has been reported but functional analyses to evaluate its significance in regulating ocular immunity are currently lacking. We compared the relative contribution of eye and gut commensals in regulating the ocular susceptibility to Pseudomonas aeruginosa-induced keratitis. We find that in health, the presence of microbiota strengthened the ocular innate immune barrier by significantly increasing the concentrations of immune effectors in the tear film, including secretory IgA and complement proteins. Consistent with this view, Swiss Webster (SW) mice that are typically resistant to P. aeruginosa-induced keratitis become susceptible due to the lack of microbiota. This was exemplified by increased corneal bacterial burden and elevated pathology of the germ free (GF) mice when compared to the conventionally maintained SW mice. The protective immunity was found to be dependent on both eye and gut microbiota with the eye microbiota having a moderate, but significant impact on the resistance to infection. These events were IL-1ß-dependent as corneal IL-1ß levels were decreased in the infected GF and antibiotic-treated mice when compared to the SPF controls, and neutralization of IL-1ß increased the ocular bacterial burden in the SPF mice. Monocolonizing GF mice with Coagulase Negative Staphylococcus sp. isolated from the conjunctival swabs was sufficient to restore resistance to infection. Cumulatively, these data underline a previously unappreciated role for microbiota in regulating susceptibility to ocular keratitis. We predict that these results will have significant implications for contact lens wearers, where alterations in the ocular commensal communities may render the ocular surface vulnerable to infections.
RESUMO
The association between inflammation and endoplasmic reticulum (ER) stress has been observed in many diseases. However, if and how chronic inflammation regulates the unfolded protein response (UPR) and alters ER homeostasis in general, or in the context of chronic disease, remains unknown. Here, we show that, in the setting of obesity, inflammatory input through increased inducible nitric oxide synthase (iNOS) activity causes S-nitrosylation of a key UPR regulator, IRE1α, which leads to a progressive decline in hepatic IRE1α-mediated XBP1 splicing activity in both genetic (ob/ob) and dietary (high-fat diet-induced) models of obesity. Finally, in obese mice with liver-specific IRE1α deficiency, reconstitution of IRE1α expression with a nitrosylation-resistant variant restored IRE1α-mediated XBP1 splicing and improved glucose homeostasis in vivo. Taken together, these data describe a mechanism by which inflammatory pathways compromise UPR function through iNOS-mediated S-nitrosylation of IRE1α, which contributes to defective IRE1α activity, impaired ER function, and prolonged ER stress in obesity.
Assuntos
Proteínas de Ligação a DNA/genética , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Óxidos de Nitrogênio/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA , Fatores de Transcrição/genética , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , Glucose/metabolismo , Homeostase , Inflamação/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Obesos , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-BoxRESUMO
Aberrant stress and inflammatory responses are key factors in the pathogenesis of obesity and metabolic dysfunction, and the double-stranded RNA-dependent kinase (PKR) has been proposed to play an important role in integrating these pathways. Here, we report the formation of a complex between PKR and TAR RNA-binding protein (TRBP) during metabolic and obesity-induced stress, which is critical for the regulation of eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and c-Jun N-terminal kinase (JNK) activation. We show that TRBP phosphorylation is induced in the setting of metabolic stress, leading to PKR activation. Suppression of hepatic TRBP reduced inflammation, JNK activity, and eIF2α phosphorylation and improved systemic insulin resistance and glucose metabolism, while TRBP overexpression exacerbated the impairment in glucose homeostasis in obese mice. These data indicate that the association between PKR and TRBP integrates metabolism with translational control and inflammatory signaling and plays important roles in metabolic homeostasis and disease.
Assuntos
Inflamação/metabolismo , Obesidade/metabolismo , Proteínas de Ligação a RNA/metabolismo , eIF-2 Quinase/metabolismo , Animais , Fator de Iniciação 2 em Eucariotos/biossíntese , Glucose/metabolismo , Humanos , Inflamação/genética , Inflamação/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Obesos , Complexos Multiproteicos/genética , Obesidade/genética , Obesidade/patologia , Fosforilação , Proteínas de Ligação a RNA/genética , Estresse Fisiológico , eIF-2 Quinase/genéticaRESUMO
As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a different charge state than the target precursor, and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably, these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent, low level background fragmentation. Finally, as a proof-of-concept 10-plex experiment, we compared phosphopeptide levels from five murine brains to five livers. In total, the SPS-MS3 method quantified 38â¯247 phosphopeptides, corresponding to 11â¯000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide, this equates to more than 628â¯000 binary comparisons collected in less than 48 h.
Assuntos
Química Encefálica , Cromatografia Líquida de Alta Pressão/métodos , Fígado/química , Espectrometria de Massas/métodos , Fosfopeptídeos/análise , Proteômica/métodos , Animais , Cromatografia de Fase Reversa/métodos , Masculino , Camundongos , FosforilaçãoRESUMO
Targeted proteomics assays such as those measuring end points in activity assays are sensitive and specific but often lack in throughput. In an effort to significantly increase throughput, a comparison was made between the traditional approach which utilizes an internal standard and the multiplexing approach which relies on isobaric tagging. A kinase activity assay was used for proof of concept, and experiments included three biological replicates for every condition. Results from the two approaches were highly similar with the multiplexing showing greater throughput. Two novel 6-plex isobaric tags were added for a total of three 6-plex experiments (18-plex) in a single run. Next, three mass variants of the target peptide were labeled with the three isobaric tags giving nine 6-plex reactions for 54-plex quantitation in a single run. Since the multiplexing approach allows all samples to be combined prior to purification and acquisition, the 54-plex approach resulted in a significant reduction in purification resources (time, reagents, etc.) and a ~50-fold improvement in acquisition throughput. We demonstrate the 54-plex assay in several ways including measuring inhibition of PKA activity in MCF7 cell lysates for a panel of nine compounds.
Assuntos
Neoplasias da Mama/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/análise , Proteômica/métodos , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Células Tumorais CultivadasRESUMO
Protein phosphorylation is an important and ubiquitous post-translational modification in eukaryotic biological systems. The KAYAK (Kinase ActivitY Assay for Kinome profiling) assay measures the phosphorylation rates of dozens of peptide substrates simultaneously, directly from cell lysates. Here, we simplified the assay by removing the phosphopeptide enrichment step, increasing throughput while maintaining similar data quality. We term this new method, direct-KAYAK, because kinase activities were measured directly from reaction mixtures after desalting. In addition, new peptides were included to profile additional kinase pathways and redundant substrate peptides were removed. Finally, the method is now performed in 96-well plate format using a benchtop orbitrap mass spectrometer and the Pinpoint software package for improved data analysis. We applied the new high-throughput method to measure IC(50) values for kinases involved in monocyte-to-macrophage differentiation, a process important for inflammation and the immune response.
Assuntos
Ensaios Enzimáticos/métodos , Espectrometria de Massas/métodos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50 , Peptídeos/química , Peptídeos/metabolismo , SoftwareRESUMO
Meiosis divides the chromosome number of the cell in half by having two rounds of chromosome segregation follow a single round of chromosome duplication. The first meiotic division is unique in that homologous pairs of sister chromatids segregate to opposite poles. Recent work in budding and fission yeast has shown that the cell cycle kinase, Cdc7-Dbf4, is required for many meiosis-specific chromosomal functions necessary for proper disjunction at meiosis I. This work reveals another role for Cdc7 in meiosis as a gene-specific regulator of the global transcription factor, Ndt80, which is required for exit from pachytene and entry into the meiotic divisions in budding yeast. Cdc7-Dbf4 promotes NDT80 transcription by relieving repression mediated by a complex of Sum1, Rfm1, and a histone deacetylase, Hst1. Sum1 exhibits meiosis-specific Cdc7-dependent phosphorylation, and mass spectrometry analysis reveals a dynamic and complex pattern of phosphorylation events, including four constitutive cyclin-dependent kinase (Cdk1) sites and 11 meiosis-specific Cdc7-Dbf4-dependent sites. Analysis of various phosphorylation site mutants suggests that Cdc7 functions with both Cdk1 and the meiosis-specific kinase Ime2 to control this critical transition point during meiosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Meiose , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Sirtuína 2/genética , Sirtuína 2/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the terminal step in methanogenesis using coenzyme B (CoBSH) as the two-electron donor to reduce methyl-coenzyme M (methyl-SCoM) to form methane and the heterodisulfide, CoBS-SCoM. The active site of MCR contains an essential redox-active nickel tetrapyrrole cofactor, coenzyme F(430), which is active in the Ni(I) state (MCR(red1)). Several catalytic mechanisms have been proposed for methane synthesis that mainly differ in whether an organometallic methyl-Ni(III) or a methyl radical is the first catalytic intermediate. A mechanism was recently proposed in which methyl-Ni(III) undergoes homolysis to generate a methyl radical (Li, X., Telser, J., Kunz, R. C., Hoffman, B. M., Gerfen, G., and Ragsdale, S. W. (2010) Biochemistry 49, 6866-6876). Discrimination among these mechanisms requires identification of the proposed intermediates, none of which have been observed with native substrates. Apparently, intermediates form and decay too rapidly to accumulate to detectible amounts during the reaction between methyl-SCoM and CoBSH. Here, we describe the reaction of methyl-SCoM with a substrate analogue (CoB(6)SH) in which the seven-carbon heptanoyl moiety of CoBSH has been replaced with a hexanoyl group. When MCR(red1) is reacted with methyl-SCoM and CoB(6)SH, methanogenesis occurs 1000-fold more slowly than with CoBSH. By transient kinetic methods, we observe decay of the active Ni(I) state coupled to formation and subsequent decay of alkyl-Ni(III) and organic radical intermediates at catalytically competent rates. The kinetic data also revealed substrate-triggered conformational changes in active Ni(I)-MCR(red1). Electron paramagnetic resonance (EPR) studies coupled with isotope labeling experiments demonstrate that the radical intermediate is not tyrosine-based. These observations provide support for a mechanism for MCR that involves methyl-Ni(III) and an organic radical as catalytic intermediates. Thus, the present study provides important mechanistic insights into the mechanism of this key enzyme that is central to biological methane formation.
Assuntos
Mesna/análogos & derivados , Methanobacteriaceae/enzimologia , Oxirredutases/metabolismo , Fosfotreonina/análogos & derivados , Acetilação , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Mesna/metabolismo , Metano/metabolismo , Methanobacteriaceae/metabolismo , Fosfotreonina/química , Fosfotreonina/metabolismo , Espectrofotometria , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step of methane formation, in which methyl-coenzyme M (2-methylthioethanesulfonate, methyl-SCoM) is reduced with coenzyme B (N-(7-mercaptoheptanoyl)threonine phosphate, CoBSH) to form methane and the heterodisulfide CoBS-SCoM. The active dimeric form of MCR contains two Ni(I)-F(430) prosthetic groups, one in each monomer. This report describes studies of the reaction of the active Ni(I) state of MCR (MCR(red1)) with BES (2-bromoethanesulfonate) and CoBSH or its analogue, CoB(6)SH (N-(6-mercaptohexanoyl)threonine phosphate), by transient kinetic measurements using EPR and UV-visible spectroscopy and by global fits of the data. This reaction is shown to lead to the formation of three intermediates, the first of which is assigned as an alkyl-Ni(III) species that forms as the active Ni(I)-MCR(red1) state of the enzyme decays. Subsequently, a radical (MCR(BES) radical) is formed that was characterized by multifrequency electron paramagnetic resonance (EPR) studies at X- ( approximately 9 GHz), Q- ( approximately 35 GHz), and D- ( approximately 130 GHz) bands and by electron-nuclear double resonance (ENDOR) spectroscopy. The MCR(BES) radical is characterized by g-values at 2.00340 and 1.99832 and includes a strongly coupled nonexchangeable proton with a hyperfine coupling constant of 50 MHz. Based on transient kinetic measurements, the formation and decay of the radical coincide with a species that exhibits absorption peaks at 426 and 575 nm. Isotopic substitution, multifrequency EPR, and ENDOR spectroscopic experiments rule out the possibility that MCR(BES) is a tyrosyl radical and indicate that if a tyrosyl radical is formed during the reaction, it does not accumulate to detectable levels. The results provide support for a hybrid mechanism of methanogenesis by MCR that includes both alkyl-Ni and radical intermediates.
Assuntos
Ácidos Alcanossulfônicos/metabolismo , Methanobacteriaceae/química , Oxirredutases/metabolismo , Ácidos Alcanossulfônicos/química , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Methanobacteriaceae/enzimologia , Modelos QuímicosRESUMO
A preference for homologs over sister chromatids in homologous recombination is a fundamental difference in meiotic versus mitotic cells. In budding yeast, the bias for interhomolog recombination in meiosis requires the Dmc1 recombinase and the meiosis-specific kinase Mek1, which suppresses engagement of sister chromatids by the mitotic recombinase Rad51. Here, a combination of proteomic, biochemical, and genetic approaches has identified an additional role for Mek1 in inhibiting the activity of the Rad51 recombinase through phosphorylation of its binding partner, Rad54. Rad54 phosphorylation of threonine 132 attenuates complex formation with Rad51, and a negative charge at this position reduces Rad51 function in vitro and in vivo. Thus, Mek1 phosphorylation provides a dynamic means of controlling recombination partner choice in meiosis in two ways: (1) it reduces Rad51 activity through inhibition of Rad51/Rad54 complex formation, and (2) it suppresses Rad51-mediated strand invasion of sister chromatids via a Rad54-independent mechanism.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , MAP Quinase Quinase 1/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Quebras de DNA de Cadeia Dupla , DNA Helicases , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Immunoblotting , MAP Quinase Quinase 1/genética , Espectrometria de Massas , Meiose , Mutação , Fosforilação , Ligação Proteica , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética , Treonina/metabolismoRESUMO
Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry-based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.