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Prostate cancer is generally considered an immunologically "cold" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to "heat up" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-ß type II receptor, bolstering its activity in the TGF-ß-rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-ß-rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2-expressing cancer cell line-derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.
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Neoplasias de la Próstata , Receptores Quiméricos de Antígenos , Masculino , Humanos , Ratones , Animales , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva , Neoplasias de la Próstata/patología , Linfocitos T , Factor de Crecimiento Transformador beta/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Microambiente Tumoral , Oxidorreductasas/metabolismoRESUMEN
BACKGROUND: Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings. METHODS: Wild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section. RESULTS: Wild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622-2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127-2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0-18% CV across consecutive tissue sections and 5-20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625). CONCLUSIONS: This work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.
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Mass spectrometry-based targeted proteomics employs heavy isotope-labeled proteins or peptides as standards to improve accuracy and precision. The input sample amount is often determined by the total quantity of endogenous proteins or peptides, as defined by spectrophotometric assays, before the heavy-isotope standards are spiked into the samples. Errors in spectrophotometric measurements, which may be due to low sensitivity or chemical or biological interference, have a direct impact on the quantitative mass spectrometry results. Currently used targeted proteomics workflows cannot identify or correct deviations that arise from differences in the input sample amount. We have developed a workflow, global extraction from parallel reaction monitoring (PRM), to identify and quantify thousands of background peptides that are inherently acquired by PRM experiments. These background peptides were used to identify differences in the input sample amount and to reduce this variance by intensity-based, post-acquisition normalization. This approach was then applied to a xenograft study to improve the quantification of human proteins in the presence of mouse tissue contamination. In addition, these background peptides also provided a direct source of quality control metrics related to sample handling and preparation.
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Péptidos , Proteómica , Animales , Espectrometría de Masas , Ratones , Proteínas , Control de CalidadRESUMEN
The human gammaherpesvirus Epstein-Barr virus (EBV) (human herpesvirus 4 [HHV4]) infects most adults and is an important contributor to the development of many types of lymphoid and epithelial cancers. Essential contributions of viral genes to viral replication are known, but the potential contributions of cell genes are less well delineated. A key player is the viral protein Zta (BZLF1, ZEBRA, or Z). This sequence-specific DNA-binding protein can disrupt EBV latency by driving the transcription of target genes and by interacting with the EBV lytic origin of replication. Here, we used an unbiased proteomics approach to identify the Zta-interactome in cells derived from Burkitt's lymphoma. Isolating Zta and associated proteins from Burkitt's lymphoma cells undergoing EBV replication, followed by tandem mass tag (TMT) mass spectrometry, resulted in the identification of 39 viral and cellular proteins within the Zta interactome. An association of Zta with the cellular protein NFATc2 was validated in independent experiments. Furthermore, the ability of Zta to attenuate the activity of an NFAT-dependent promoter was shown, which suggests a functional consequence for the association. The expression of Zta is itself regulated through NFAT activity, suggesting that Zta may contribute to a feedback loop that would limit its own expression, thus aiding viral replication by preventing the known toxic effects of Zta overexpression.IMPORTANCE Epstein-Barr virus infects most people across the world and causes several kinds of cancer. Zta is an important viral protein that makes the virus replicate by binding to its DNA and turning on the expression of some genes. We used a sensitive, unbiased approach to isolate and identify viral and cellular proteins that physically interact with Zta. This revealed 39 viral and cellular proteins. We found that one protein, termed NFATc2, was already known to be important for a very early step in viral replication. We identify that once this step has occurred, Zta reduces the effectiveness of NFATc2, and we suggest that this is important to prevent cells from dying before viral replication is complete and the mature virus is released from the cells.
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Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Transactivadores/genética , Transactivadores/metabolismo , Replicación Viral/genética , Linfoma de Burkitt , Línea Celular , Proteínas de Unión al ADN/metabolismo , Genes Virales , Humanos , Factores de Transcripción NFATC/metabolismo , Regiones Promotoras Genéticas , Proteómica , Proteínas Virales/genética , Proteínas Virales/metabolismo , Latencia del VirusRESUMEN
Mass spectrometry-based protein quantitation is currently used to measure therapeutically relevant protein biomarkers in CAP/CLIA setting to predict likely responses of known therapies. Selected reaction monitoring (SRM) is the method of choice due to its outstanding analytical performance. However, data-independent acquisition (DIA) is now emerging as a proteome-scale clinical assay. We evaluated the ability of DIA to profile the patient-specific proteomes of sample-limited tumor biopsies and to quantify proteins of interest in a targeted fashion using formalin-fixed, paraffin-embedded (FFPE) tumor biopsies ( n = 12) selected from our clinical laboratory. DIA analysis on the tumor biopsies provided 3713 quantifiable proteins including actionable biomarkers currently in clinical use, successfully separated two gastric cancers from colorectal cancer specimen solely on the basis of global proteomic profiles, and identified subtype-specific proteins with prognostic or diagnostic value. We demonstrate the potential use of DIA-based quantitation to inform therapeutic decision-making using TUBB3, for which clinical cutoff expression levels have been established by SRM. Comparative analysis of DIA-based proteomic profiles and mRNA expression levels found positively and negatively correlated protein-gene pairs, a finding consistent with previously reported results from fresh-frozen tumor tissues.
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Espectrometría de Masas/métodos , Neoplasias/química , Patología Molecular/métodos , Proteoma/análisis , Biomarcadores de Tumor/análisis , Biopsia , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , Humanos , Neoplasias/patología , Adhesión en Parafina , Proteómica/métodos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patología , Fijación del TejidoRESUMEN
Deletions and chromosome re-arrangements are common features of cancer cells. We have established a new two-component system reporting on epigenetic silencing or deletion of an actively transcribed gene adjacent to a double-strand break (DSB). Unexpectedly, we find that a targeted DSB results in a minority (<10%) misrepair event of kilobase deletions encompassing the DSB site and transcribed gene. Deletions are reduced upon RNaseH1 over-expression and increased after knockdown of the DNA:RNA helicase Senataxin, implicating a role for DNA:RNA hybrids. We further demonstrate that the majority of these large deletions are dependent on the 3' flap endonuclease XPF. DNA:RNA hybrids were detected by DNA:RNA immunoprecipitation in our system after DSB generation. These hybrids were reduced by RNaseH1 over-expression and increased by Senataxin knock-down, consistent with a role in deletions. Overall, these data are consistent with DNA:RNA hybrid generation at the site of a DSB, mis-processing of which results in genome instability in the form of large deletions.
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Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , ARN Helicasas/fisiología , Línea Celular Tumoral , ADN/genética , Roturas del ADN de Doble Cadena , ADN Helicasas/fisiología , Proteínas de Unión al ADN/genética , Endonucleasas/metabolismo , Inestabilidad Genómica , Humanos , Enzimas Multifuncionales , ARN , ARN Helicasas/metabolismo , Eliminación de Secuencia/genéticaRESUMEN
The Cardiomyopathy-associated gene 5 (Cmya5) encodes myospryn, a large tripartite motif (TRIM)-related protein found predominantly in cardiac and skeletal muscle. Cmya5 is an expression biomarker for a number of diseases affecting striated muscle and may also be a schizophrenia risk gene. To further understand the function of myospryn in striated muscle, we searched for additional myospryn paralogs. Here we identify a novel muscle-expressed TRIM-related protein minispryn, encoded by Fsd2, that has extensive sequence similarity with the C-terminus of myospryn. Cmya5 and Fsd2 appear to have originated by a chromosomal duplication and are found within evolutionarily-conserved gene clusters on different chromosomes. Using immunoaffinity purification and mass spectrometry we show that minispryn co-purifies with myospryn and the major cardiac ryanodine receptor (RyR2) from heart. Accordingly, myospryn, minispryn and RyR2 co-localise at the junctional sarcoplasmic reticulum of isolated cardiomyocytes. Myospryn redistributes RyR2 into clusters when co-expressed in heterologous cells whereas minispryn lacks this activity. Together these data suggest a novel role for the myospryn complex in the assembly of ryanodine receptor clusters in striated muscle.
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Proteínas Portadoras/genética , Clonación Molecular/métodos , Proteínas Musculares/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Chlorocebus aethiops , Cromatografía de Afinidad , Duplicación Cromosómica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Espectrometría de Masas , Ratones , Proteínas Musculares/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break.
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Roturas del ADN de Cadena Simple , Histonas/metabolismo , Nucleosomas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ribosa/metabolismo , Animales , Línea Celular , Pollos , Cromatina/metabolismo , Cromosomas/metabolismo , ADN/metabolismo , Reparación del ADN , Humanos , Modelos Moleculares , Poli(ADP-Ribosa) Polimerasas/química , Dominios ProteicosRESUMEN
The working model to describe the mechanisms used to replicate the cancer-associated virus Epstein-Barr virus (EBV) is partly derived from comparisons with other members of the Herpes virus family. Many genes within the EBV genome are homologous across the herpes virus family. Published transcriptome data for the EBV genome during its lytic replication cycle show extensive transcription, but the identification of the proteins is limited. We have taken a global proteomics approach to identify viral proteins that are expressed during the EBV lytic replication cycle. We combined an enrichment method to isolate cells undergoing EBV lytic replication with SILAC-labeling coupled to mass-spectrometry and identified viral and host proteins expressed during the OPEN ACCESS Pathogens 2015, 4 740 EBV lytic replication cycle. Amongst the most frequently identified viral proteins are two components of the DNA replication machinery, the single strand DNA binding protein BALF2, DNA polymerase accessory protein BMRF1 and both subunits of the viral ribonucleoside-diphosphate reductase enzyme (BORF2 and BaRF1). An additional 42 EBV lytic cycle proteins were also detected. This provides proteomic identification for many EBV lytic replication cycle proteins and also identifies post-translational modifications.
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We employ stable-isotope labeling and quantitative mass spectrometry to track histone methylation stability. We show that H3 trimethyl K9 and K27 are slow to be established on new histones and slow to disappear from old histones, with half-lives of multiple cell divisions. By contrast, the transcription-associated marks K4me3 and K36me3 turn over far more rapidly, with half-lives of 6.8 h and 57 h, respectively. Inhibition of demethylases increases K9 and K36 methylation, with K9 showing the largest and most robust increase. We interpret different turnover rates in light of genome-wide localization data and transcription-dependent nucleosome rearrangements proximal to the transcription start site.
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Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Cromatina/química , Cromatina/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Marcaje Isotópico , Lisina/química , Metilación , Estabilidad ProteicaRESUMEN
Cellular senescence, an irreversible cell cycle arrest induced by a diversity of stimuli, has been considered as an innate tumor suppressing mechanism with implications and applications in cancer therapy. Using a targeted proteomics approach, we show that fibroblasts induced into senescence by expression of oncogenic Ras exhibit a decrease of global acetylation on all core histones, consistent with formation of senescence-associated heterochromatic foci. We also detected clear increases in repressive markers (e.g. >50% elevation of H3K27me2/3) along with decreases in histone marks associated with increased transcriptional expression/elongation (e.g. H3K36me2/3). Despite the increases in repressive marks of chromatin, 179 loci (of 2206 total) were found to be upregulated by global quantitative proteomics. The changes in the cytosolic proteome indicated an upregulation of mitochondrial proteins and downregulation of proteins involved in glycolysis. These alterations in primary metabolism are opposite to the well-known Warburg effect observed in cancer cells. This study significantly improves our understanding of stress-induced senescence and provides a potential application for triggering it in antiproliferative strategies that target the primary metabolism in cancer cells.
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Senescencia Celular/genética , Glucólisis/fisiología , Histonas/metabolismo , Proteínas Mitocondriales/biosíntesis , Neoplasias/metabolismo , Oncogenes , Proteómica/métodos , Proteínas ras/genética , Acetilación , Puntos de Control del Ciclo Celular , Línea Celular , Proliferación Celular , Cromatina/metabolismo , Cromatografía Liquida/métodos , Citosol/metabolismo , Regulación hacia Abajo , Fibroblastos , Glucólisis/genética , Humanos , Neoplasias/genética , Neoplasias/patología , Proteoma/metabolismo , Espectrometría de Masas en Tándem/métodos , Transcripción Genética , Regulación hacia ArribaRESUMEN
Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Mapas de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Sitios de Unión , Proteínas del Citoesqueleto , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Oligopéptidos/análisis , Fosfoproteínas/genética , Fosforilación , Fosfotirosina/química , Fosfotirosina/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Proteómica , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transducción de Señal , Familia-src Quinasas/genéticaRESUMEN
We have developed a targeted method to quantify all combinations of methylation on an H3 peptide containing lysines 27 and 36 (H3K27-K36). By using stable isotopes that separately label the histone backbone and its methylations, we tracked the rates of methylation and demethylation in myeloma cells expressing high vs. low levels of the methyltransferase MMSET/WHSC1/NSD2. Following quantification of 99 labeled H3K27-K36 methylation states across time, a kinetic model converged to yield 44 effective rate constants qualifying each methylation and demethylation step as a function of the methylation state on the neighboring lysine. We call this approach MS-based measurement and modeling of histone methylation kinetics (M4K). M4K revealed that, when dimethylation states are reached on H3K27 or H3K36, rates of further methylation on the other site are reduced as much as 100-fold. Overall, cells with high MMSET have as much as 33-fold increases in the effective rate constants for formation of H3K36 mono- and dimethylation. At H3K27, cells with high MMSET have elevated formation of K27me1, but even higher increases in the effective rate constants for its reversal by demethylation. These quantitative studies lay bare a bidirectional antagonism between H3K27 and H3K36 that controls the writing and erasing of these methylation marks. Additionally, the integrated kinetic model was used to correctly predict observed abundances of H3K27-K36 methylation states within 5% of that actually established in perturbed cells. Such predictive power for how histone methylations are established should have major value as this family of methyltransferases matures as drug targets.
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N-Metiltransferasa de Histona-Lisina/química , Histonas/química , Lisina/química , Proteínas Represoras/química , Bioquímica/métodos , Línea Celular , Técnicas Químicas Combinatorias , Epigenómica , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Cinética , Espectrometría de Masas/métodos , Metilación , Metiltransferasas/química , Proteínas Represoras/genéticaRESUMEN
A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.
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Isoformas de Proteínas/análisis , Isoformas de Proteínas/química , Proteoma/análisis , Proteoma/química , Proteómica/métodos , Empalme Alternativo , Línea Celular , Senescencia Celular/genética , Daño del ADN , Bases de Datos de Proteínas , Proteína HMGA1a/análisis , Proteína HMGA1b/análisis , Células HeLa , Humanos , Fenotipo , Procesamiento Proteico-Postraduccional , Proteolisis , Proteómica/instrumentaciónRESUMEN
The multiple myeloma SET domain (MMSET) protein is overexpressed in multiple myeloma (MM) patients with the translocation t(4;14). Although studies have shown the involvement of MMSET/Wolf-Hirschhorn syndrome candidate 1 in development, its mode of action in the pathogenesis of MM is largely unknown. We found that MMSET is a major regulator of chromatin structure and transcription in t(4;14) MM cells. High levels of MMSET correlate with an increase in lysine 36 methylation of histone H3 and a decrease in lysine 27 methylation across the genome, leading to a more open structural state of the chromatin. Loss of MMSET expression alters adhesion properties, suppresses growth, and induces apoptosis in MM cells. Consequently, genes affected by high levels of MMSET are implicated in the p53 pathway, cell cycle regulation, and integrin signaling. Regulation of many of these genes required functional histone methyl-transferase activity of MMSET. These results implicate MMSET as a major epigenetic regulator in t(4;14)+ MM.
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Cromosomas Humanos Par 14/genética , Cromosomas Humanos Par 4/genética , Metilación de ADN , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Mieloma Múltiple/genética , Proteínas Represoras/genética , Translocación Genética/genética , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Adhesión Celular , Ciclo Celular , Movimiento Celular , Proliferación Celular , Cromatina/genética , Inmunoprecipitación de Cromatina , Epigenómica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Applying high-throughput Top-Down MS to an entire proteome requires a yet-to-be-established model for data processing. Since Top-Down is becoming possible on a large scale, we report our latest software pipeline dedicated to capturing the full value of intact protein data in automated fashion. For intact mass detection, we combine algorithms for processing MS1 data from both isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, which are then linked to their fragment ions for database searching using ProSight. Automated determination of human keratin and tubulin isoforms is one result. Optimized for the intricacies of whole proteins, new software modules visualize proteome-scale data based on the LC retention time and intensity of intact masses and enable selective detection of PTMs to automatically screen for acetylation, phosphorylation, and methylation. Software functionality was demonstrated using comparative LC-MS data from yeast strains in addition to human cells undergoing chemical stress. We further these advances as a key aspect of realizing Top-Down MS on a proteomic scale.
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Espectrometría de Masas , Proteómica , Algoritmos , Secuencia de Aminoácidos , Proteínas Fúngicas/análisis , Células HeLa , Histonas/análisis , Histonas/genética , Humanos , Queratinas/análisis , Queratinas/genética , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Proteómica/instrumentación , Proteómica/métodos , Programas Informáticos , Estatmina/análisis , Estatmina/genética , Tubulina (Proteína)/análisis , Tubulina (Proteína)/genéticaRESUMEN
We employ a stable isotope strategy wherein both histones and their methylations are labeled in synchronized human cells. This allows us to differentiate between old and new methylations on pre-existing versus newly synthesized histones. The strategy is implemented on K79 methylation in an isoform-specific manner for histones H3.1, H3.2, and H3.3. Although levels of H3.3K79 monomethylation are higher than that of H3.2/H3.1, the rate of establishing the K79 methylation is the same for all three isoforms. Surprisingly, we find that pre-existing "old" histones continue to be K79-monomethylated and -dimethylated at a rate equal to the newly synthesized histones. These observations imply that some degree of positional "scrambling" of K79 methylation occurs through the cell cycle.
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Ciclo Celular/fisiología , Cromatina/metabolismo , Histonas/metabolismo , Células HeLa , Humanos , MetilaciónRESUMEN
Fibroblast growth factor receptors (FGFR) are cell surface tyrosine kinases that function in cell proliferation and differentiation. Aberrant FGFR signaling occurs in diverse cancers due to gene amplification, but the associated oncogenic mechanisms are poorly understood. Using a proteomics approach, we identified signal transducers and activators of transcription-3 (STAT3) as a receptor-binding partner that is mediated by Tyr(677) phosphorylation on FGFR. Binding to activated FGFR was essential for subsequent tyrosine phosphorylation and nuclear translocation of STAT3, along with activation of its downstream target genes. Tyrosine phosphorylation of STAT3 was also dependent on concomitant FGFR-dependent activity of SRC and JAK kinases. Lastly, tyrosine (but not serine) phosphorylation of STAT3 required amplified FGFR protein expression, generated either by enforced overexpression or as associated with gene amplification in cancer cells. Our findings show that amplified FGFR expression engages the STAT3 pathway, and they suggest therapeutic strategies to attack FGFR-overexpressing cancers.
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Regulación Neoplásica de la Expresión Génica , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Miembro 25 de Receptores de Factores de Necrosis Tumoral/metabolismo , Línea Celular , Línea Celular Tumoral , Células HeLa , Humanos , Janus Quinasa 1/metabolismo , Fosforilación , Unión Proteica , Transducción de Señal , Transcripción Genética , Tirosina/química , Familia-src Quinasas/metabolismoRESUMEN
Activation of signal transduction by the receptor tyrosine kinase, fibroblast growth factor receptor (FGFR), results in a cascade of protein-protein interactions that rely on the occurrence of specific tyrosine phosphorylation events. One such protein recruited to the activated receptor complex is the nonreceptor tyrosine kinase, Src, which is involved in both initiation and termination of further signaling events. To gain a further understanding of the tyrosine phosphorylation events that occur during FGF signaling, with a specific focus on those that are dependent on Src family kinase (SFK) activity, we have applied SILAC combined with chemical inhibition of SFK activity to search for phosphorylation events that are dependent on SFK activity in FGF stimulated cells. In addition, we used a more targeted approach to carry out high coverage phosphopeptide mapping of one Src substrate protein, the multifunctional adaptor Dok1, and to identify SFK-dependent Dok1 binding partners. From these analyses we identify 80 SFK-dependent phosphorylation events on 40 proteins. We further identify 18 SFK-dependent Dok1 interactions and 9 SFK-dependent Dok1 phosphorylation sites, 6 of which had not previously been known to be SFK-dependent.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factor 2 de Crecimiento de Fibroblastos/química , Humanos , Inmunoprecipitación , Marcaje Isotópico , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Péptidos/química , Péptidos/metabolismo , Fosfoproteínas/química , Fosforilación , Unión Proteica , Proteoma/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Familia-src Quinasas/químicaRESUMEN
Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC-MS to drive high-throughput top-down proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation and detection of fragment ions with <10 ppm mass accuracy for highly specific database searching using tailored software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines prefractionated by their molecular mass using a gel-based sieving system.