RESUMEN
The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes. Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1-cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate. PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.
Asunto(s)
Mitosis , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Proteína Quinasa CDC2/metabolismo , Segregación Cromosómica , Secuencia Conservada , Ciclina B/metabolismo , Activación Enzimática , Células HeLa , Holoenzimas/metabolismo , Humanos , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Fosforilación , Proteína Fosfatasa 2/química , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Transducción de SeñalRESUMEN
The Rac activator Tiam1 is required for adherens junction (AJ) maintenance, and its depletion results in AJ disassembly. Conversely, the oncoprotein Src potently induces AJ disassembly and epithelial-mesenchymal transition (EMT). Here, we show that Tiam1 is phosphorylated on Y384 by Src. This occurs predominantly at AJs, is required for Src-induced AJ disassembly and cell migration, and creates a docking site on Tiam1 for Grb2. We find that Tiam1 is associated with ERK. Following recruitment of the Grb2-Sos1 complex, ERK becomes activated and triggers the localized degradation of Tiam1 at AJs, likely involving calpain proteases. Furthermore, we demonstrate that, in human tumors, Y384 phosphorylation positively correlates with Src activity, and total Tiam1 levels are inversely correlated. Thus, our data implicate Tiam1 phosphorylation and consequent degradation in Src-mediated EMT and resultant cell motility and establish a paradigm for regulating local concentrations of Rho-GEFs.
Asunto(s)
Uniones Adherentes/enzimología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Procesamiento Proteico-Postraduccional , Familia-src Quinasas/metabolismo , Animales , Calpaína/deficiencia , Calpaína/genética , Calpaína/metabolismo , Línea Celular , Movimiento Celular , Clonación Molecular , Perros , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína Adaptadora GRB2/metabolismo , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Mutación , Invasividad Neoplásica , Neoplasias/enzimología , Neoplasias/patología , Proteína Oncogénica pp60(v-src)/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-yes/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteína SOS1/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Factores de Tiempo , Transfección , Tirosina , Dominios Homologos src , Familia-src Quinasas/genéticaRESUMEN
We developed a murine model of CNS disease to obtain a better understanding of the pathogenesis of CNS involvement in pre-B-cell acute lymphoblastic leukemia (ALL). Semiquantitative proteomic discovery-based approaches identified unique expression of asparaginyl endopeptidase (AEP), intercellular adhesion molecule 1 (ICAM1), and ras-related C3 botulinum toxin substrate 2 (RAC2), among others, in an invasive pre-B-cell line that produced CNS leukemia in NOD-SCID mice. Targeting RAC2 significantly inhibited in vitro invasion and delayed disease onset in mice. Induced expression of RAC2 in cell lines with low/absent expression of AEP and ICAM1 did not result in an invasive phenotype or murine CNS disease. Flow cytometric analysis identified an enriched population of blast cells expressing ICAM1/lymphocyte function associated antigen-1 (LFA-1)/CD70 in the CD10(+)/CD19(+) fraction of bone marrow aspirates obtained from relapsed compared with normal controls and those with primary disease. CD10(+)/CD19(+) fractions obtained from relapsed patients also express RAC2 and give rise to CNS disease in mice. Our data suggest that combinations of processes are involved in the pathogenesis of CNS disease in pre-B-cell ALL, support a model in which CNS disease occurs as a result of external invasion, and suggest that targeting the processes of adhesion and invasion unique to pre-B cells may prevent recurrences within the CNS.
Asunto(s)
Neoplasias del Sistema Nervioso Central/fisiopatología , Cisteína Endopeptidasas/genética , Molécula 1 de Adhesión Intercelular/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/fisiopatología , Proteínas de Unión al GTP rac/genética , Animales , Adhesión Celular/fisiología , Línea Celular Tumoral , Membrana Celular/fisiología , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/patología , Niño , Cisteína Endopeptidasas/metabolismo , Modelos Animales de Enfermedad , Regulación Leucémica de la Expresión Génica/fisiología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Proteómica , Proteínas de Unión al GTP rac/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
RATIONALE: Mapping sites of wild-type SUMO modification is a challenging endeavour. Here we postulate that a combination of chemical derivatistation and collision-induced dissociation (CID) could be used to generate SUMO remnant diagnostic ions to aid both detection of these isopeptides and increase the analytical value of the product ion spectra required to characterize the nature and position of modification. METHODS: SUMO(2/3)ylated proteins were digested with trypsin to generate isopeptides bearing TGG and QTGG isotags. The resulting digests were then dimethyl labelled followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) utilising CID in a data-dependent acquisition on a QSTAR XL. Product ion spectra were interrogated for the presence of iso-N-terminal fragment ions in addition to backbone sequence ions. The ability to diagnostically detect these isopeptides was tested by generation of co-XICs of the iso-N-terminal fragments in a semi-complex background. RESULTS: Dimethyl labelling facilitated the robust detection of a1', b2' & b3' (TGG isotag) and a1', b2' & b4' (QTGG isotag) ions. The abundance of both N-terminal and iso-N-terminal fragment ions, supported by dimethyl labelling, facilitated the generation of information-rich product ion spectra of these isopeptides to aid confident site assignment. Moreover, the diagnostic nature of the combined XICs of the iso-N-terminal fragments supported detection of the isopeptide signals from a semi-complex background. CONCLUSIONS: A combination of dimethyl labelling and CID does indeed lead to the generation of SUMO remnant isopeptide product ion spectra which are more analytically rich. This enables an improvement in characterization of both the isotag and backbone sequences and the site of modification. The diagnostic value of iso-N-terminal fragment ions allows for post-acquisition XIC interrogation to detect putative isopeptides of interest.
Asunto(s)
Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Mapeo Peptídico , Sumoilación , Espectrometría de Masas en Tándem , Tripsina/químicaRESUMEN
RATIONALE: Identification of sites of protein SUMOylation is of great importance due its functional diversity within the cell. To date, most approaches to this problem rely on site-directed mutagenesis and/or highly specialised mass spectrometry approaches. We present a novel alternative approach to the site mapping of SUMOylation using trypsin and elastase digestion, routine mass spectrometry and an unbiased isotag database searching strategy. METHODS: SUMOylated protein samples were digested with a number of enzymes and the resulting peptides separated using liquid chromatography. Analysis was carried out on both linear ion trap Orbitrap and quadrupole-time-of-flight (Q-TOF)-based mass spectrometers equipped with electrospray ionisation. The data files were subsequently searched using the Mascot algorithm with multiple variable tag modifications corresponding to SUMO-derived fragments. The utility of this approach was demonstrated with di-SUMO 2, di-SUMO 3, SUMO 1-RanGap(418-587) 1 and an enriched population of SUMOylated proteins. RESULTS: Unbiased database searches led to the identification of a number of analytically useful isotags ranging in length from two to four residues. Isopeptide fragments were generated including QTGG (di-SUMO-2/3), TGG (di-SUMO-2/3) and GG (SUMO-1). The method was validated by successfully mapping a number of sites of SUMO modification on SUMO-modified proteins enriched from a cell lysate. CONCLUSIONS: This combination of relaxed enzyme specificity, shortened isotag generation and unbiased database searching enabled confident identification of novel analytically useful SUMOylated isopeptides without a requirement for mutagenesis.
Asunto(s)
Bases de Datos de Proteínas , Fragmentos de Péptidos/metabolismo , Proteínas/metabolismo , Sumoilación , Secuencia de Aminoácidos , Cromatografía Liquida , Biología Computacional , Células HEK293 , Humanos , Lisina/química , Lisina/metabolismo , Datos de Secuencia Molecular , Elastasa Pancreática/metabolismo , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Proteínas/análisis , Proteínas/química , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Tripsina/metabolismoRESUMEN
AIMS: Accurate determination of HER-2 status is critical to identify patients for whom trastuzumab treatment will be of benefit. Although the recommended primary method of evaluation is immunohistochemistry, numerous reports of variability in interpretation have raised uncertainty about the reliability of results. Recent guidelines have suggested that image analysis could be an effective tool for achieving consistent interpretation, and this study aimed to assess whether this technology has potential as a diagnostic support tool. METHODS AND RESULTS: Across a cohort of 275 cases, image analysis could accurately classify HER-2 status, with 91% agreement between computer-aided classification and the pathology review. Assessment of the continuity of membranous immunoreactivity in addition to intensity of reactivity was critical to distinguish between negative and equivocal cases and enabled image analysis to report a lower referral rate of cases for confirmatory fluorescence in situ hybridization (FISH) testing. An excellent concordance rate of 95% was observed between FISH and the automated review across 136 informative cases. CONCLUSIONS: This study has validated that image analysis can robustly and accurately evaluate HER-2 status in immunohistochemically stained tissue. Based on these findings, image analysis has great potential as a diagnostic support tool for pathologists and biomedical scientists, and may significantly improve the standardization of HER-2 testing by providing a quantitative reference method for interpretation.
Asunto(s)
Neoplasias de la Mama/química , Neoplasias de la Mama/diagnóstico , Procesamiento de Imagen Asistido por Computador/métodos , Receptor ErbB-2/análisis , Algoritmos , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Estudios de Cohortes , Diagnóstico por Computador , Femenino , Genes erbB-2 , Humanos , Procesamiento de Imagen Asistido por Computador/normas , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ , TrastuzumabRESUMEN
AIM: To assess a new HER2 fluorescence in situ hybridization (FISH) test and report on multicentre intrasite and intersite variation. METHODS AND RESULTS: HER2 results were scored from 45 breast cancers in eight laboratories using the Kreatech Poseidon HER2 FISH probe (Kreatech Diagnostics, Amsterdam, the Netherlands). Overall, 80.9% of cores were successfully analysed. Mean intrasite variation for HER2 ratio assessment was low (4.74%). Intersite variation in ratio was in line with previous reports (11.9+/-0.8%) for both reference and non-reference laboratories; only one laboratory displayed significantly higher intersite variation (P=0.009) than the remaining seven laboratories. The overall incidence of misclassification of cores was <1.3%, demonstrating an excellent level of concordance (>98.7%) across all eight laboratories, irrespective of whether they were 'reference' or 'routine diagnostic' laboratories. CONCLUSIONS: The Kreatech Poseidon HER2 FISH test is robust and reproducible. Highly quantitatively reproducible FISH results were obtained from eight 'diagnostic' and 'reference' laboratories; however, continued quality assessments are essential to good performance.
Asunto(s)
Neoplasias de la Mama/genética , Perfilación de la Expresión Génica/normas , Genes erbB-2 , Hibridación Fluorescente in Situ/normas , Patología Clínica/normas , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Hibridación Fluorescente in Situ/métodos , Patología Clínica/métodos , Reproducibilidad de los Resultados , Análisis de Matrices Tisulares , Reino UnidoRESUMEN
BACKGROUND: Previous studies comparing quantitative proteomics and microarray data have generally found poor correspondence between the two. We hypothesised that this might in part be because the different assays were targeting different parts of the expressed genome and might therefore be subjected to confounding effects from processes such as alternative splicing. RESULTS: Using a genome database as a platform for integration, we combined quantitative protein mass spectrometry with Affymetrix Exon array data at the level of individual exons. We found significantly higher degrees of correlation than have been previously observed (r = 0.808). The study was performed using cell lines in equilibrium in order to reduce a major potential source of biological variation, thus allowing the analysis to focus on the data integration methods in order to establish their performance. CONCLUSION: We conclude that part of the variation observed when integrating microarray and proteomics data may occur as a consequence both of the data analysis and of the high granularity to which studies have until recently been limited. The approach opens up the possibility for the first time of considering combined microarray and proteomics datasets at the level of individual exons and isoforms, important given the high proportion of alternative splicing observed in the human genome.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Mapeo Peptídico/métodos , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodos , Sitios de Empalme de ARN/genética , Algoritmos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Integración de SistemasRESUMEN
The fission yeast scaffold molecule Sid4 anchors the septum initiation network to the spindle pole body (SPB, centrosome equivalent) to control mitotic exit events. A second SPB-associated scaffold, Cut12, promotes SPB-associated Cdk1-cyclin B to drive mitotic commitment. Signals emanating from each scaffold have been assumed to operate independently to promote two distinct outcomes. We now find that signals from Sid4 contribute to the Cut12 mitotic commitment switch. Specifically, phosphorylation of Sid4 by NIMAFin1 reduces Sid4 affinity for its SPB anchor, Ppc89, while also enhancing Sid4's affinity for casein kinase 1δ (CK1δ). The resulting phosphorylation of Sid4 by the newly docked CK1δ recruits Chk2Cds1 to Sid4. Chk2Cds1 then expels the Cdk1-cyclin B antagonistic phosphatase Flp1/Clp1 from the SPB. Flp1/Clp1 departure can then support mitotic commitment when Cdk1-cyclin B activation at the SPB is compromised by reduction of Cut12 function. Such integration of signals emanating from neighboring scaffolds shows how centrosomes/SPBs can integrate inputs from multiple pathways to control cell fate.
Asunto(s)
Centrosoma/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Cuerpos Polares del Huso/metabolismo , Sitios de Unión , Quinasa Idelta de la Caseína/genética , Quinasa Idelta de la Caseína/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Mutación , Quinasa 1 Relacionada con NIMA/genética , Quinasa 1 Relacionada con NIMA/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/genética , Transducción de Señal , Cuerpos Polares del Huso/genética , Factores de TiempoAsunto(s)
Biomarcadores de Tumor/genética , Reordenamiento Génico , Linfoma de Células B Grandes Difuso/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-6/genética , Biomarcadores de Tumor/metabolismo , Humanos , Incidencia , Irlanda/epidemiología , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/epidemiología , Linfoma de Células B Grandes Difuso/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Estudios Retrospectivos , Regulación hacia ArribaRESUMEN
Angiogenesis is intimately related to the growth and progression of tumours and must be induced to facilitate growth beyond a minimum size. It has been implicated in the development of metastases and survival in breast carcinoma. VEGF is a cytokine that plays an important role in angiogenesis. Its expression is increased in solid tumours during induction of angiogenesis and it has been implicated as a prognostic marker in patients with node negative breast carcinoma. We studied VEGF expression, in a series of patients with node positive breast carcinoma and examined histopathological parameters of the tumour and the prognostic value of VEGF expression. Specimens from 108 cases of node positive breast cancer were stained for VEGF using an antibody suitable for use on formalin fixed tissue. VEGF staining was cytoplasmic and was scored by intensity and the percent positive cells. Patients with positive VEGF staining (n=48) were compared with patients with negative VEGF staining (n=60). Demographic criteria were similar in both groups. Only one (12%) patient with lobular carcinoma and one (14%) patient with medullary carcinoma expressed VEGF compared with 46 (49%) patients with ductal carcinoma (NOS). DCIS was present in 60 tumours. There was a strong correlation between staining in DCIS and the adjacent invasive tumours. There was no significant association between VEGF staining and T stage, tumour size or the number of positive lymph nodes. VEGF expression had no prognostic significance either for disease-free or overall survival in patients with node positive disease. This study failed to support a role for VEGF as a prognostic marker in patients with node positive breast carcinoma.
Asunto(s)
Neoplasias de la Mama/metabolismo , Factores de Crecimiento Endotelial/metabolismo , Linfocinas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/terapia , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Carcinoma Intraductal no Infiltrante/metabolismo , Carcinoma Intraductal no Infiltrante/patología , Carcinoma Intraductal no Infiltrante/terapia , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Ganglios Linfáticos/patología , Persona de Mediana Edad , Invasividad Neoplásica , Neoplasias Ductales, Lobulillares y Medulares/metabolismo , Neoplasias Ductales, Lobulillares y Medulares/patología , Neoplasias Ductales, Lobulillares y Medulares/terapia , Pronóstico , Receptores de Estrógenos/metabolismo , Tasa de Supervivencia , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Non-coding RNAs (ncRNAs) are frequent and prevalent across the taxa. Although individual non-coding loci have been assigned a function, most are uncharacterized. Their global biological significance is unproven and remains controversial. Here we investigate the role played by ncRNAs in the stress response of Schizosaccharomyces pombe. We integrate global proteomics and RNA sequencing data to identify a systematic programme in which elevated antisense RNA arising both from ncRNAs and from 3'-overlapping convergent gene pairs is directly associated with substantial reductions in protein levels throughout the genome. We describe an extensive array of ncRNAs with trans associations that have the potential to influence multiple pathways. Deletion of one such locus reduces levels of atf1, a transcription factor downstream of the stress-activated mitogen-activated protein kinase (MAPK) pathway, and alters sensitivity to oxidative stress. These non-coding transcripts therefore regulate specific stress responses, adding unanticipated information-processing capacity to the MAPK signalling system.
Asunto(s)
ARN de Hongos/genética , ARN no Traducido/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Estrés Fisiológico/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Sistemas de Lectura Abierta/genética , Presión Osmótica , Estrés Oxidativo/genética , Proteoma/metabolismo , ARN sin Sentido/metabolismo , ARN de Hongos/metabolismo , ARN no Traducido/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transcriptoma/genéticaRESUMEN
Protein modification by ubiquitination and SUMOylation occur throughout the cell and are responsible for numerous cellular functions such as apoptosis, DNA replication and repair, and gene transcription. Current methods for the identification of such modifications using mass spectrometry predominantly rely upon tryptic isopeptide tag generation followed by database searching with in vitro genetic mutation of SUMO routinely required. We have recently described a novel approach to ubiquitin and SUMO modification detection based upon the diagnostic a' and b' ions released from the isopeptide tags upon collision-induced dissociation of reductively methylated Ubl isopeptides (RUbI) using formaldehyde. Here, we significantly extend those studies by combining data-independent acquisition (DIA) with alternative labeling reagents to improve diagnostic ion coverage and enable relative quantification of modified peptides from both MS and MS/MS signals. Model synthetic ubiquitin and SUMO-derived isopeptides were labeled with mTRAQ reagents (Δ0, Δ4, and Δ8) and subjected to LC-MS/MS with SWATH acquisition. Novel diagnostic ions were generated upon CID, which facilitated the selective detection of these modified peptides. Simultaneous MS-based and MS/MS-based relative quantification was demonstrated for both Ub and SUMO-derived isopeptides across three channels in a background of mTRAQ-labeled Escherichia coli digest.
Asunto(s)
Modelos Moleculares , Péptidos/química , Proteínas Ubiquitinadas/química , Métodos Analíticos de la Preparación de la Muestra , Cromatografía Líquida de Alta Presión , Técnicas Electroquímicas , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Marcaje Isotópico , Espectrometría de Masas , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptidos/metabolismo , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Procesamiento de Señales Asistido por Computador , Sumoilación , Espectrometría de Masas en Tándem , Proteínas Ubiquitinadas/metabolismoRESUMEN
Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2' and a1' ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1' and b2' diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers. á
Asunto(s)
Péptidos/análisis , Espectrometría de Masas en Tándem/métodos , Ubiquitina/química , Proteínas Ubiquitinadas/química , Secuencia de Aminoácidos , Glicina/análisis , Glicilglicina/análisis , Humanos , Metilación , Datos de Secuencia Molecular , Oxidación-Reducción , Poliubiquitina/químicaRESUMEN
BACKGROUND: Activation of the Cdk1/cyclin B complex, also known as mitosis-promoting factor (MPF), drives commitment to mitosis. Interphase MPF is inhibited through phosphorylation of Cdk1 by Wee1-related kinases. Because Cdc25 phosphatases remove this phosphate, Cdc25 activity is an essential part of the switch that drives cells into mitosis. The generation of a critical "trigger" of active MPF promotes a positive feedback loop that employs Polo kinase to boost Cdc25 activity and inhibit Wee1, thereby ensuring that mitotic commitment is a bistable switch. Mutations in the spindle pole body (SPB) component Cut12 suppress otherwise lethal deficiencies in Cdc25. RESULTS: Cut12 harbors a bipartite protein phosphatase 1 (PP1) docking domain. Mutation of either element alone suppressed the temperature-dependent lethality of cdc25.22, whereas simultaneous ablation of both allowed cells to divide in the complete absence of Cdc25. Late G2 phase phosphorylation between the two elements by MPF and the NIMA kinase Fin1 blocked PP1(Dis2) recruitment, thereby promoting recruitment of Polo to Cut12 and the SPB and elevating global Polo kinase activity throughout the cell. CONCLUSIONS: PP1 recruitment to Cut12 sets a threshold for Polo's feedback-loop activity that locks the cell in interphase until Cdc25 pushes MPF activity through this barrier to initiate mitosis. We propose that events on the SPB (and, by inference, the centrosome) integrate inputs from diverse signaling networks to generate a coherent decision to divide that is appropriate for the particular environmental context of each cell. PP1 recruitment sets one or more critical thresholds for single or multiple local events within this switch.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Fosfoproteínas/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Centrosoma/enzimología , Factor Promotor de Maduración/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Quinasa 1 Relacionada con NIMA , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/genética , Proteínas Quinasas/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Mitotic exit integrates the reversal of the phosphorylation events initiated by mitotic kinases with a controlled cytokinesis event that cleaves the cell in two. The mitotic exit network (MEN) of budding yeast regulates both processes, whereas the fission yeast equivalent, the septum initiation network (SIN), controls only the execution of cytokinesis. The components and architecture of the SIN and MEN are highly conserved. At present, it is assumed that the functions of the core SIN-MEN components are restricted to their characterized roles at the end of mitosis. We now show that the NDR (nuclear Dbf2-related) kinase component of the fission yeast SIN, Sid2-Mob1, acts independently of the other known SIN components in G2 phase of the cell cycle to control the timing of mitotic commitment. Sid2-Mob1 promotes mitotic commitment by directly activating the NIMA (Never In Mitosis)-related kinase Fin1. Fin1's activation promotes its own destruction, thereby making Fin1 activation a transient feature of G2 phase. This spike of Fin1 activation modulates the activity of the Pom1/Cdr1/Cdr2 geometry network towards Wee1.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Citocinesis , Proteínas del Citoesqueleto/metabolismo , Mitosis , Proteínas Nucleares/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Transducción de Señal , Proteínas de Ciclo Celular/genética , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Fase G2 , Mutación , Proteínas Nucleares/genética , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/antagonistas & inhibidores , Proteínas de Schizosaccharomyces pombe/genética , Serina , Factores de TiempoRESUMEN
We present the first investigation into the utility of porous graphitic carbon (PGC) as a stationary phase in proteomic workflows involving complex samples. PGC offers chemical and physical robustness and is capable of withstanding extremes of pH and higher temperatures than traditional stationary phases, without the likelihood of catastrophic failure. In addition, unlike separations driven by ion exchange mechanisms, there is no requirement for high levels of non-volatile salts such as potassium chloride in the elution buffers, which must be removed prior to LC-MS analysis. Here we present data which demonstrate that PGC affords excellent peptide separation in a complex whole cell lysate digest sample, with good orthogonality to a typical low pH reversed-phase system. As strong cation exchange (SCX) is currently the most popular first dimension for 2D peptide separations, we chose to compare the performance of a PGC and SCX separation as the first dimension in a comprehensive 2D-LC-MS/MS workflow. A significant increase, in the region of 40%, in peptide identifications is reported with off-line PGC fractionation compared to SCX. Around 14,000 unique peptides were identified at an estimated false discovery rate of 1% (n=3 replicates) from starting material constituting only 100 µg of protein extract.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Grafito/química , Fragmentos de Péptidos/análisis , Proteómica/métodos , Cationes/química , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Fragmentos de Péptidos/química , Porosidad , Proteínas de Schizosaccharomyces pombe/análisis , Proteínas de Schizosaccharomyces pombe/química , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Vigorous chromosome movements driven by cytoskeletal assemblies are a widely conserved feature of sexual differentiation to facilitate meiotic recombination. In fission yeast, this process involves the dramatic conversion of arrays of cytoplasmic microtubules (MTs), generated from multiple MT organizing centers (MTOCs), into a single radial MT (rMT) array associated with the spindle pole body (SPB), the major MTOC during meiotic prophase. The rMT is then dissolved upon the onset of meiosis I when a bipolar spindle emerges to conduct chromosome segregation. Structural features and molecular mechanisms that govern these dynamic MT rearrangements are poorly understood. RESULTS: Electron tomography of the SPBs showed that the rMT emanates from a newly recognized amorphous structure, which we term the rMTOC. The rMTOC, which resides at the cytoplasmic side of the SPB, is highly enriched in γ-tubulin reminiscent of the pericentriolar material of higher eukaryotic centrosomes. Formation of the rMTOC depends on Hrs1/Mcp6, a meiosis-specific SPB component that is located at the rMTOC. At the onset of meiosis I, Hrs1/Mcp6 is subject to strict downregulation by both proteasome-dependent degradation and phosphorylation leading to complete inactivation of the rMTOC. This ensures rMT dissolution and bipolar spindle formation. CONCLUSIONS: Our study reveals the molecular basis for the transient generation of a novel MTOC, which triggers a program of MT rearrangement that is required for meiotic differentiation.
Asunto(s)
Núcleo Celular/fisiología , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Huso Acromático/metabolismo , Meiosis , Microscopía Fluorescente , Tubulina (Proteína)/metabolismoRESUMEN
BACKGROUND: Most protein mass spectrometry (MS) experiments rely on searches against a database of known or predicted proteins, limiting their ability as a gene discovery tool. RESULTS: Using a search against an in silico translation of the entire human genome, combined with a series of annotation filters, we identified 346 putative novel peptides [False Discovery Rate (FDR)<5%] in a MS dataset derived from two human breast epithelial cell lines. A subset of these were then successfully validated by a different MS technique. Two of these correspond to novel isoforms of Heterogeneous Ribonuclear Proteins, while the rest correspond to novel loci. CONCLUSIONS: MS technology can be used for ab initio gene discovery in human data, which, since it is based on different underlying assumptions, identifies protein-coding genes not found by other techniques. As MS technology continues to evolve, such approaches will become increasingly powerful.