RESUMEN
As spaceflight becomes more common with commercial crews, blood-based measures of crew health can guide both astronaut biomedicine and countermeasures. By profiling plasma proteins, metabolites, and extracellular vesicles/particles (EVPs) from the SpaceX Inspiration4 crew, we generated "spaceflight secretome profiles," which showed significant differences in coagulation, oxidative stress, and brain-enriched proteins. While >93% of differentially abundant proteins (DAPs) in vesicles and metabolites recovered within six months, the majority (73%) of plasma DAPs were still perturbed post-flight. Moreover, these proteomic alterations correlated better with peripheral blood mononuclear cells than whole blood, suggesting that immune cells contribute more DAPs than erythrocytes. Finally, to discern possible mechanisms leading to brain-enriched protein detection and blood-brain barrier (BBB) disruption, we examined protein changes in dissected brains of spaceflight mice, which showed increases in PECAM-1, a marker of BBB integrity. These data highlight how even short-duration spaceflight can disrupt human and murine physiology and identify spaceflight biomarkers that can guide countermeasure development.
Asunto(s)
Coagulación Sanguínea , Barrera Hematoencefálica , Encéfalo , Homeostasis , Estrés Oxidativo , Vuelo Espacial , Animales , Humanos , Encéfalo/metabolismo , Barrera Hematoencefálica/metabolismo , Ratones , Coagulación Sanguínea/fisiología , Masculino , Secretoma/metabolismo , Ratones Endogámicos C57BL , Vesículas Extracelulares/metabolismo , Proteómica/métodos , Biomarcadores/metabolismo , Biomarcadores/sangre , Femenino , Adulto , Proteínas Sanguíneas/metabolismo , Persona de Mediana Edad , Leucocitos Mononucleares/metabolismo , Proteoma/metabolismoRESUMEN
The liver is the only solid organ capable of regenerating itself to regain 100% of its mass and function after liver injury and/or partial hepatectomy (PH). This exceptional property represents a therapeutic opportunity for severe liver disease patients. However, liver regeneration (LR) might fail due to poorly understood causes. Here, we have investigated the regulation of liver proteome and phosphoproteome at a short time after PH (9 h), to depict a detailed mechanistic background of the early LR phase. Furthermore, we analyzed the dynamic changes of the serum proteome and metabolome of healthy living donor liver transplant (LDLT) donors at different time points after surgery. The molecular profiles from both analyses were then correlated. Insulin and FXR-FGF15/19 signaling were stimulated in mouse liver after PH, leading to the activation of the main intermediary kinases (AKT and ERK). Besides, inhibition of the hippo pathway led to an increased expression of its target genes and of one of its intermediary proteins (14-3-3 protein), contributing to cell proliferation. In association with these processes, metabolic reprogramming coupled to enhanced mitochondrial activity cope for the energy and biosynthetic requirements of LR. In human serum of LDLT donors, we identified 56 proteins and 13 metabolites statistically differential which recapitulate some of the main cellular processes orchestrating LR in its early phase. These results provide mechanisms and protein mediators of LR that might prove useful for the follow-up of the regenerative process in the liver after PH as well as preventing the occurrence of complications associated with liver resection.
Asunto(s)
Regeneración Hepática , Trasplante de Hígado , Ratones , Animales , Humanos , Regeneración Hepática/genética , Trasplante de Hígado/métodos , Proteoma/genética , Proteoma/metabolismo , Donadores Vivos , Hígado/cirugía , Hígado/metabolismoRESUMEN
Absolute quantification measurements (copies per cell) of peptide major histocompatibility complex (pMHC) antigens are necessary to inform targeted immunotherapy drug design; however, existing methods for absolute quantification have critical limitations. Here, we present a platform termed SureQuant-IsoMHC, utilizing a series of pMHC isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multipoint calibration curve to determine endogenous pMHC concentrations for a panel of 18 tumor antigens. We apply SureQuant-IsoMHC to measure changes in expression of our target panel in a melanoma cell line treated with a MEK inhibitor and translate this approach to estimate antigen concentrations in melanoma tumor biopsies.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/análisis , Bencimidazoles/farmacología , Antígenos de Histocompatibilidad Clase I/inmunología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Melanoma/inmunología , Presentación de Antígeno/efectos de los fármacos , Antígenos de Neoplasias/efectos de los fármacos , Antígenos de Neoplasias/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoterapia , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Células Tumorales CultivadasRESUMEN
Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry-based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed "SureQuant pTyr," coupling low input pTyr enrichment with a panel of isotopically labeled internal standard peptides to guide data acquisition of low-abundance tyrosine phosphopeptides. SureQuant pTyr allowed for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, improving the robustness and usability of targeted mass spectrometry assays. We established the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient-specific oncogenic-driving mechanisms. While in some cases pTyr profiles aligned with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlighted instances of new insights gained using pTyr characterization and emphasized the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enables pTyr-based measurements for applications in precision medicine. SIGNIFICANCE: SureQuant pTyr is a mass spectrometry-based targeted method that enables sensitive and selective targeted quantitation of several hundred low-abundance tyrosine phosphorylated peptides commonly dysregulated in cancer, including oncogenic signaling networks.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/análisis , Transducción de Señal , Tirosina/metabolismo , Células A549 , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Cromatografía Liquida/métodos , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Receptores ErbB/metabolismo , Humanos , Espectrometría de Masas/métodos , Fosfopéptidos/análisis , Fosfopéptidos/metabolismo , Fosforilación , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Drug-induced compensatory signaling and subsequent rewiring of the signaling pathways that support cell proliferation and survival promote the development of acquired drug resistance in tumors. Here, we sought to analyze the adaptive kinase response in cancer cells after distinct treatment with agents targeting human epidermal growth factor receptor 2 (HER2), specifically those that induce either only temporary cell cycle arrest or, alternatively, apoptosis in HER2-overexpressing cancers. We compared trastuzumab, ARRY380, the combination thereof, and a biparatopic, HER2-targeted designed ankyrin repeat protein (DARPin; specifically, 6L1G) and quantified the phosphoproteome by isobaric tagging using tandem mass tag liquid chromatography/tandem mass spectrometry (TMT LC-MS/MS). We found a specific signature of persistently phosphorylated tyrosine peptides after the nonapoptotic treatments, which we used to distinguish between different treatment-induced cancer cell fates. Next, we analyzed the activation of serine/threonine and tyrosine kinases after treatment using a bait peptide chip array and predicted the corresponding active kinases. Through a combined system-wide analysis, we identified a common adaptive kinase response program that involved the activation of focal adhesion kinase 1 (FAK1), protein kinase C-δ (PRKCD), and Ephrin (EPH) family receptors. These findings reveal potential targets to prevent adaptive resistance to HER2-targeted therapies.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas Quinasas/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Trastuzumab/farmacología , Antineoplásicos Inmunológicos/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromatografía Liquida , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Fosfoproteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteoma/metabolismo , Receptor ErbB-2/metabolismo , Espectrometría de Masas en TándemRESUMEN
Despite extensive study of the EGF receptor (EGFR) signaling network, the immediate posttranslational changes that occur in response to growth factor stimulation remain poorly characterized; as a result, the biological mechanisms underlying signaling initiation remain obscured. To address this deficiency, we have used a mass spectrometry-based approach to measure system-wide phosphorylation changes throughout the network with 10-s resolution in the 80 s after stimulation in response to a range of eight growth factor concentrations. Significant changes were observed on proteins far downstream in the network as early as 10 s after stimulation, indicating a system capable of transmitting information quickly. Meanwhile, canonical members of the EGFR signaling network fall into clusters with distinct activation patterns. Src homology 2 domain containing transforming protein (Shc) and phosphoinositol 3-kinase (PI3K) phosphorylation levels increase rapidly, but equilibrate within 20 s, whereas proteins such as Grb2-associated binder-1 (Gab1) and SH2-containing tyrosine phosphatase (SHP2) show slower, sustained increases. Proximity ligation assays reveal that Shc and Gab1 phosphorylation patterns are representative of separate timescales for physical association with the receptor. Inhibition of phosphatases with vanadate reveals site-specific regulatory mechanisms and also uncovers primed activating components in the network, including Src family kinases, whose inhibition affects only a subset of proteins within the network. The results presented highlight the complexity of signaling initiation and provide a window into exploring mechanistic hypotheses about receptor tyrosine kinase (RTK) biology.
Asunto(s)
Mama/enzimología , Receptores ErbB/fisiología , Transducción de Señal/fisiología , Mama/citología , División Celular , Línea Celular , Medio de Cultivo Libre de Suero/farmacología , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Receptores ErbB/agonistas , Femenino , Humanos , Complejos Multiproteicos , Fosfoproteínas Fosfatasas/fisiología , Fosfoproteínas/análisis , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Factores de Tiempo , Familia-src Quinasas/fisiologíaRESUMEN
Tumor protein phosphorylation analysis may provide insight into intracellular signaling networks underlying tumor behavior, revealing diagnostic, prognostic or therapeutic information. Human tumors collected by The Cancer Genome Atlas program potentially offer the opportunity to characterize activated networks driving tumor progression, in parallel with the genetic and transcriptional landscape already documented for these tumors. However, a critical question is whether cellular signaling networks can be reliably analyzed in surgical specimens, where freezing delays and spatial sampling disparities may potentially obscure physiologic signaling. To quantify the extent of these effects, we analyzed the stability of phosphotyrosine (pTyr) sites in ovarian and colon tumors collected under conditions of controlled ischemia and in the context of defined intratumoral sampling. Cold-ischemia produced a rapid, unpredictable, and widespread impact on tumor pTyr networks within 5 minutes of resection, altering up to 50% of pTyr sites by more than 2-fold. Effects on adhesion and migration, inflammatory response, proliferation, and stress response pathways were recapitulated in both ovarian and colon tumors. In addition, sampling of spatially distinct colon tumor biopsies revealed pTyr differences as dramatic as those associated with ischemic times, despite uniform protein expression profiles. Moreover, intratumoral spatial heterogeneity and pTyr dynamic response to ischemia varied dramatically between tumors collected from different patients. Overall, these findings reveal unforeseen phosphorylation complexity, thereby increasing the difficulty of extracting physiologically relevant pTyr signaling networks from archived tissue specimens. In light of this data, prospective tumor pTyr analysis will require appropriate sampling and collection protocols to preserve in vivo signaling features.
Asunto(s)
Fosfotirosina/metabolismo , Artefactos , Hipoxia de la Célula , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Neoplasias Ováricas/metabolismo , Fosforilación , Estudios Prospectivos , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
Attempts to characterize cellular behaviors with static, univariate measurements cannot fully capture biological complexity and lead to an inadequate interpretation of cellular processes. Significant biological insight can be gleaned by considering the contribution of dynamic protein post-translational modifications (PTMs) utilizing systems-level quantitative analysis. High-resolution mass spectrometry coupled with computational modeling of dynamic signal-response relationships is a powerful tool to reveal PTM-mediated regulatory networks. Recent advances using this approach have defined network kinetics of growth factor signaling pathways, identified systems level responses to cytotoxic perturbations, elucidated kinase-substrate relationships, and unraveled the dynamics of PTM cross-talk. Innovations in multiplex measurement capacity, PTM annotation accuracy, and computational integration of datasets promise enhanced resolution of dynamic PTM networks and further insight into biological intricacies.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Animales , Humanos , Espectrometría de Masas , Fosforilación , Proteínas/química , Transducción de SeñalRESUMEN
Production of haploid gametes from diploid progenitor cells is mediated by a specialized cell division, meiosis, where two divisions, meiosis I and II, follow a single S phase. Errors in progression from meiosis I to meiosis II lead to aneuploid and polyploid gametes, but the regulatory mechanisms controlling this transition are poorly understood. Here, we demonstrate that the conserved kinase Ime2 regulates the timing and order of the meiotic divisions by controlling translation. Ime2 coordinates translational activation of a cluster of genes at the meiosis I-meiosis II transition, including the critical determinant of the meiotic chromosome segregation pattern CLB3. We further show that Ime2 mediates translational control through the meiosis-specific RNA-binding protein Rim4. Rim4 inhibits translation of CLB3 during meiosis I by interacting with the 5' untranslated region (UTR) of CLB3. At the onset of meiosis II, Ime2 kinase activity rises and triggers a decrease in Rim4 protein levels, thereby alleviating translational repression. Our results elucidate a novel developmentally regulated translational control pathway that establishes the meiotic chromosome segregation pattern.
Asunto(s)
Segregación Cromosómica/genética , Regulación Fúngica de la Expresión Génica , Meiosis/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 5'/genética , Péptidos y Proteínas de Señalización Intracelular , Familia de Multigenes/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Numb is an endocytic adaptor protein that regulates the endocytosis and trafficking of transmembrane receptors including Notch, E-cadherin, and integrins. Vertebrate Numb is alternatively spliced at exons 3 and 9 to give rise to four protein isoforms. Expression of these isoforms varies at different developmental stages, and although the function of Numb isoforms containing exon 3 has been studied, the role of exon 9 inclusion has not been shown. Here we use affinity purification and tandem mass spectrometry to identify Numb associated proteins, including novel interactions with REPS1, BMP2K, and BCR. In vitro binding measurements indicated exon 9-independent Numb interaction with REPS1 and Eps15 EH domains. Selected reaction monitoring mass spectrometry was used to quantitatively compare the proteins associated with the p72 and p66 Numb isoforms, which differ by the exon 9 region. This showed that significantly more EPS15 and three AP-2 subunit proteins bound Numb isoforms containing exon 9. The EPS15 preference for exon 9-containing Numb was confirmed in intact cells by using a proximity ligation assay. Finally, we used multiplexed selected reaction monitoring mass spectrometry to assess the dynamic regulation of Numb association with endocytic proteins. Numb hyper-phosphorylation resulted in disassociation of Numb endocytic complexes, while inhibition of endocytosis did not alter Numb association with the AP-2 complex but altered recruitment of EPS15, REPS1, and BMP2K. Hence, quantitative mass spectrometric analysis of Numb protein-protein interactions has provided new insights into the assembly and regulation of protein complexes important in development and cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Portadoras/genética , Endocitosis/genética , Factores de Transcripción de Tipo Kruppel/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas c-bcr/genética , Proteínas Adaptadoras Transductoras de Señales/aislamiento & purificación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Unión al Calcio , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Cromatografía Liquida , Exones , Regulación de la Expresión Génica , Células HEK293 , Humanos , Factores de Transcripción de Tipo Kruppel/aislamiento & purificación , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcr/aislamiento & purificación , Proteínas Proto-Oncogénicas c-bcr/metabolismo , Transducción de Señal , Espectrometría de Masas en Tándem/métodos , TransfecciónRESUMEN
Aberrations in epidermal growth factor receptor (EGFR/ErbB1) are the most common oncogenic alterations in glioblastoma multiforme (GBM), the most common primary brain tumor. Interactions between wild-type (wt) and mutant EGFRs and their subsequent activation are of biologic and potential therapeutic importance in GBMs. We recently showed that in situ proximity ligation assay (PLA) allows for quantitative evaluation of EGFR dimerization and activation in intact cells. Using this in situ platform, we show the aberrant homo-/heterodimeric properties of EGFRvIII and EGFRc958 mutants, the two most common EGFR mutants in GBMs. In addition, dimer phosphoactivation status could be detected by PLA with superior signal-noise ratio (>17-fold) and sensitivity (>16-fold) than immunofluorescence-based phospho-EGFR measurements. Dimer activation analysis indicated quantitative activation differences of mutant dimers. These aberrant features were not overexpression dependent but appeared independent of cellular expression levels, suggesting inherent properties of the mutant receptors. Moreover, we observed in situ detection of EGFRwt-EGFRvIII heterodimerization in GBM specimens, supporting our cell line observations. Notably, currently used anti-EGFR therapeutics, such as cetuximab, matuzumab, and panitumumab, could effectively block EGFRwt dimerization and activation but did not equally impair EGFRvIII homodimers, EGFRwt-EGFRvIII, or EGFRvIII-EGFRc958 heterodimers. EGFRvIII appears to have intrinsic phosphoactivation independent of dimerization as matuzumab blockade of homodimerization had no effect on receptor phosphorylation levels. These data suggest differences in the dimerization-blocking efficacy of EGFR monoclonal antibodies as mutant EGFR dimer configurations prevalent in GBMs can evade blockade by anti-EGFR treatments. Further studies are warranted to evaluate whether this evasion contributes to poor therapeutic response or resistance.
Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Glioblastoma/enzimología , Terapia Molecular Dirigida , Proteínas Mutantes/metabolismo , Multimerización de Proteína , Animales , Anticuerpos Antineoplásicos/inmunología , Bioensayo , Células CHO , Cricetinae , Cricetulus , Activación Enzimática , Epítopos/inmunología , Receptores ErbB/inmunología , Humanos , Immunoblotting , Fosforilación , Relación Señal-RuidoRESUMEN
Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O6-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O6-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N7-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.