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1.
Int J Mol Sci ; 22(6)2021 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-33804769

RESUMEN

SARS-CoV-2 infection can cause cytokine storm and may overshoot immunity in humans; however, it remains to be determined whether virus-induced soluble mediators from infected cells are carried by exosomes as vehicles to distant organs and cause tissue damage in COVID-19 patients. We took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and COVID-19 patients. Our results revealed that tenascin-C (TNC) and fibrinogen-ß (FGB) are highly abundant in exosomes from COVID-19 patients' plasma compared with that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via the Nuclear factor-κB (NF-κB) pathway, we examined the status of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-C motif chemokine ligand 5 (CCL5) expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase compared with that from healthy subjects. Together, our results demonstrate that TNC and FGB are transported through plasma exosomes and potentially trigger pro-inflammatory cytokine signaling in cells of distant organ.


Asunto(s)
/sangre , Exosomas/química , Exosomas/genética , Fibrinógeno/metabolismo , Inflamación/metabolismo , Tenascina/metabolismo , Anciano , Línea Celular , Quimiocina CCL5/metabolismo , Exosomas/metabolismo , Exosomas/ultraestructura , Femenino , Hepatocitos/metabolismo , Humanos , Inflamación/etiología , Interleucina-6/metabolismo , Masculino , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , FN-kappa B/metabolismo , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
2.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-33669532

RESUMEN

Although understanding of the biomedical basis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is growing, the underlying pathological mechanisms remain uncertain. We recently reported a reduction in the proportion of basal oxygen consumption due to ATP synthesis by Complex V in ME/CFS patient-derived lymphoblast cell lines, suggesting mitochondrial respiratory inefficiency. This was accompanied by elevated respiratory capacity, elevated mammalian target of rapamycin complex 1 (mTORC1) signaling activity and elevated expression of enzymes involved in the TCA cycle, fatty acid ß-oxidation and mitochondrial transport. These and other observations led us to hypothesise the dysregulation of pathways providing the mitochondria with oxidisable substrates. In our current study, we aimed to revisit this hypothesis by applying a combination of whole-cell transcriptomics, proteomics and energy stress signaling activity measures using subsets of up to 34 ME/CFS and 31 healthy control lymphoblast cell lines from our growing library. While levels of glycolytic enzymes were unchanged in accordance with our previous observations of unaltered glycolytic rates, the whole-cell proteomes of ME/CFS lymphoblasts contained elevated levels of enzymes involved in the TCA cycle (p = 1.03 × 10-4), the pentose phosphate pathway (p = 0.034, G6PD p = 5.5 × 10-4), mitochondrial fatty acid ß-oxidation (p = 9.2 × 10-3), and degradation of amino acids including glutamine/glutamate (GLS p = 0.034, GLUD1 p = 0.048, GOT2 p = 0.026), branched-chain amino acids (BCKDHA p = 0.028, BCKDHB p = 0.031) and essential amino acids (FAH p = 0.036, GCDH p = 0.006). The activity of the major cellular energy stress sensor, AMPK, was elevated but the increase did not reach statistical significance. The results suggest that ME/CFS metabolism is dysregulated such that alternatives to glycolysis are more heavily utilised than in controls to provide the mitochondria with oxidisable substrates.


Asunto(s)
Síndrome de Fatiga Crónica/metabolismo , Linfocitos/metabolismo , Mitocondrias/metabolismo , Adulto , Anciano , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Redes y Vías Metabólicas , Persona de Mediana Edad , Oxidación-Reducción , Fosforilación Oxidativa , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/metabolismo , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato , Transcriptoma/genética
3.
Nat Commun ; 12(1): 1830, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758186

RESUMEN

Aminoglycoside antibiotics target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal aminoglycosides induce not only single translation errors, but also clusters of errors in full-length proteins in vivo with as many as four amino acid substitutions in a row. The downstream errors in a cluster are up to 10,000-fold more frequent than the first error and independent of the intracellular aminoglycoside concentration. The prevalence, length, and composition of error clusters depends not only on the misreading propensity of a given aminoglycoside, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a distinct class of misreading events in vivo that may provide the predominant source of proteotoxic stress at low aminoglycoside concentration, which is particularly important for the autocatalytic uptake of the drugs.


Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Factor Tu de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas/genética , Proteoma/genética , Ribosomas/metabolismo , Estrés Fisiológico/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Espectrometría de Masas , Mutación Missense , Nebramicina/análogos & derivados , Nebramicina/farmacología , Factor Tu de Elongación Peptídica/genética , Péptidos/genética , Péptidos/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteoma/efectos de los fármacos , Proteoma/metabolismo , Proteómica , Proteínas Recombinantes , Ribosomas/efectos de los fármacos , Estreptomicina/farmacología , Estrés Fisiológico/genética
4.
Nat Commun ; 12(1): 1396, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33654096

RESUMEN

Increasing numbers of protein interactions have been identified in high-throughput experiments, but only a small proportion have solved structures. Recently, sequence coevolution-based approaches have led to a breakthrough in predicting monomer protein structures and protein interaction interfaces. Here, we address the challenges of large-scale interaction prediction at residue resolution with a fast alignment concatenation method and a probabilistic score for the interaction of residues. Importantly, this method (EVcomplex2) is able to assess the likelihood of a protein interaction, as we show here applied to large-scale experimental datasets where the pairwise interactions are unknown. We predict 504 interactions de novo in the E. coli membrane proteome, including 243 that are newly discovered. While EVcomplex2 does not require available structures, coevolving residue pairs can be used to produce structural models of protein interactions, as done here for membrane complexes including the Flagellar Hook-Filament Junction and the Tol/Pal complex.


Asunto(s)
Aminoácidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evolución Molecular , Genoma Bacteriano , Mapeo de Interacción de Proteínas , Proteínas Bacterianas/química , Secuencia de Bases , Escherichia coli/genética , Células Eucariotas/metabolismo , Proteínas de la Membrana/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Proteoma/metabolismo
5.
Int J Nanomedicine ; 16: 1943-1960, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33727808

RESUMEN

Introduction: The overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is usually associated with aggressive and infiltrating breast cancer (BC) phenotype, and metastases. Functionalized silica-based nanocarriers (SiNPs) can be labeled for in vivo imaging applications and loaded with chemotherapy drugs, making possible the simultaneous noninvasive diagnosis and treatment (theranostic) for HER2-positive BC. Methods: Firstly, FITC-filled SiNPs, were engineered with two different amounts of Hc-TZ (trastuzumab half-chain) per single nanoparticle (1:2 and 1:8, SiNPs to Hc-TZ ratio), which was 99mTc-radiolabeled at histidine residues for ex vivo and in vivo biodistribution evaluations. Secondly, nanoparticles were loaded with DOX and their in vitro and ex vivo/in vivo delivery was assessed, in comparison with liposomal Doxorubicin (Caelyx). Finally, the treatment efficacy of DOX-SiNPs-TZ (1:8 Hc-TZ) was evaluated in vivo by PET and supported by MS-based proteomics profiling of tumors. Results: SiNPs-TZ (1:8 Hc-TZ) tumor uptake was significantly greater than that of SiNPs-TZ (1:2 Hc-TZ) at 6 hours post-injection (p.i.) in ex vivo biodistribution experiment. At 24 h p.i., radioactivity values remained steady. Fluorescence microscopy, confirmed the presence of radiolabeled SiNPs-TZ (1:8 Hc-TZ) within tumor even at later times. SiNPs-TZ (1:8 Hc-TZ) nanoparticles loaded with Doxorubicin (DOX-SiNPs-TZ) showed a similar DOX delivery capability than Caelyx (at 6 h p.i.), in in vitro and ex vivo assays. Nevertheless, at the end of treatment, tumor volume was significantly reduced by DOX-SiNPs-TZ (1:8 Hc-TZ), compared to Caelyx and DOX-SiNPs treatment. Proteomics study identified 88 high stringent differentially expressed proteins comparing the three treatment groups with controls. Conclusion: These findings demonstrated a promising detection specificity and treatment efficacy for our system (SiNPs-TZ, 1:8 Hc-TZ), encouraging its potential use as a new theranostic agent for HER2-positive BC lesions. In addition, proteomic profile confirmed that a set of proteins, related to tumor aggressiveness, were positively affected by targeted nanoparticles.


Asunto(s)
Neoplasias de la Mama/diagnóstico , Portadores de Fármacos/química , Nanopartículas/química , Radiofármacos/química , Receptor ErbB-2/metabolismo , Dióxido de Silicio/química , Tecnecio/química , Animales , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Endocitosis , Femenino , Fluoresceína-5-Isotiocianato/química , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Polietilenglicoles/química , Polietilenglicoles/farmacología , Polietilenglicoles/uso terapéutico , Proteoma/metabolismo , Proteómica , Radiofármacos/farmacocinética , Tecnecio/farmacocinética , Distribución Tisular/efectos de los fármacos , Tomografía Computarizada de Emisión de Fotón Único , Resultado del Tratamiento
6.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-33669748

RESUMEN

Muse cells are adult stem cells that are present in the stroma of several organs and possess an enduring capacity to cope with endogenous and exogenous genotoxic stress. In cell therapy, the peculiar biological properties of Muse cells render them a possible natural alternative to mesenchymal stromal cells (MSCs) or to in vitro-generated pluripotent stem cells (iPSCs). Indeed, some studies have proved that Muse cells can survive in adverse microenvironments, such as those present in damaged/injured tissues. We performed an evaluation of Muse cells' proteome under basic conditions and followed oxidative stress treatment in order to identify ontologies, pathways, and networks that can be related to their enduring stress capacity. We executed the same analysis on iPSCs and MSCs, as a comparison. The Muse cells are enriched in several ontologies and pathways, such as endosomal vacuolar trafficking related to stress response, ubiquitin and proteasome degradation, and reactive oxygen scavenging. In Muse cells, the protein-protein interacting network has two key nodes with a high connectivity degree and betweenness: NFKB and CRKL. The protein NFKB is an almost-ubiquitous transcription factor related to many biological processes and can also have a role in protecting cells from apoptosis during exposure to a variety of stressors. CRKL is an adaptor protein and constitutes an integral part of the stress-activated protein kinase (SAPK) pathway. The identified pathways and networks are all involved in the quality control of cell components and may explain the stress resistance of Muse cells.


Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Proteoma/metabolismo , Proteómica , Estrés Fisiológico , Línea Celular , Daño del ADN , Ontología de Genes , Humanos , Células Madre Pluripotentes Inducidas/citología , Mapas de Interacción de Proteínas , Transducción de Señal
7.
Methods Mol Biol ; 2218: 277-290, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606239

RESUMEN

Rapid innovations in core proteomic technologies and proteome-based bioinformatics fortified by recent genome sequencing allow the characterization and quantification of proteins on a global scale. These capabilities empower research to develop a more comprehensive understanding of how changes in protein expression and modification can affect complex signaling and regulatory networks. The consequences of these studies have significant implications for understanding how myriad activities are regulated in biological systems.Proteomic approaches have been applied to investigate the physiology, developmental biology, and impact of contaminants in fishes as model organisms. Here, we describe the use of label-free protein quantification and global proteome profiling to characterize eggs of different quality grades in the zebrafish .


Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Biología Computacional/métodos , Huevos , Femenino , Masculino , Proteoma/metabolismo , Proteómica/métodos
8.
Methods Mol Biol ; 2218: 291-302, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606240

RESUMEN

Protein production and degradation are tightly regulated to prevent cellular structures from accumulating damage and to allow their correct functioning. A key aspect of this regulation is the protein half-life, corresponding to the time in which half of a specific protein population is exchanged with respect to its initial state. Proteome-wide techniques to investigate protein half-lives in vivo are emerging. Recently, we have established and thoroughly tested a metabolic labeling approach using 13C lysine (Lys(6)) for measuring protein lifetimes in mice. The approach is based on the fact that different proteins will incorporate a metabolic label at a rate that is dependent on their half-life. Using amino acid pool modeling and mass spectrometry, it is possible to measure the fraction of newly synthesized proteins and determine protein half-lives. In this chapter, we show how to extend this approach to zebrafish (Danio rerio), using a commercially available fish diet based on the stable isotope labeling by amino acids in cell culture (SILAC) technology. We describe the methods for labeling animals and subsequently use mass spectrometry to determine the lifetimes of a large number of proteins. In the mass spectrometry workflow proposed here, we have implemented the BoxCar data acquisition approach for increasing sample coverage and optimize machine use. To establish the proteome library used in the BoxCar approach, we recommend performing an in-solution digestion followed by peptide fractionation through basic reversed-phase chromatography. Overall, this chapter extends the use of current proteome technologies for the quantification of protein turnover to zebrafish and similar organisms and permits the study of germline changes following specific manipulations.


Asunto(s)
Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Aminoácidos/metabolismo , Animales , Femenino , Semivida , Marcaje Isotópico/métodos , Lisina/metabolismo , Masculino , Proteoma/metabolismo , Proteómica/métodos
9.
Commun Biol ; 4(1): 225, 2021 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-33580175

RESUMEN

Serodiagnosis of SARS-CoV-2 infection is impeded by immunological cross-reactivity among the human coronaviruses (HCoVs): SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43, 229E, HKU1, and NL63. Here we report the identification of humoral immune responses to SARS-CoV-2 peptides that may enable discrimination between exposure to SARS-CoV-2 and other HCoVs. We used a high-density peptide microarray and plasma samples collected at two time points from 50 subjects with SARS-CoV-2 infection confirmed by qPCR, samples collected in 2004-2005 from 11 subjects with IgG antibodies to SARS-CoV-1, 11 subjects with IgG antibodies to other seasonal human coronaviruses (HCoV), and 10 healthy human subjects. Through statistical modeling with linear regression and multidimensional scaling we identified specific peptides that were reassembled to identify 29 linear SARS-CoV-2 epitopes that were immunoreactive with plasma from individuals who had asymptomatic, mild or severe SARS-CoV-2 infections. Larger studies will be required to determine whether these peptides may be useful in serodiagnostics.


Asunto(s)
/inmunología , Mapeo Peptídico , Péptidos/inmunología , /fisiología , Secuencia de Aminoácidos , Animales , Quirópteros , Epítopos/inmunología , Humanos , Inmunoglobulina G/metabolismo , Péptidos/química , Proteoma/metabolismo
10.
Nat Commun ; 12(1): 1244, 2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33623024

RESUMEN

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.


Asunto(s)
Diferenciación Celular , Leishmania mexicana/citología , Leishmania mexicana/enzimología , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Supervivencia Celular , Femenino , Flagelos/enzimología , Eliminación de Gen , Leishmaniasis/parasitología , Leishmaniasis/patología , Ratones Endogámicos BALB C , Modelos Biológicos , Mutación/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteoma/metabolismo , Psychodidae/parasitología
11.
Nat Commun ; 12(1): 1230, 2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33623002

RESUMEN

The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Oxigenasas de Función Mixta/metabolismo , Polisacáridos/metabolismo , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Animales , Proteínas Bacterianas/química , Proteínas Portadoras/química , Muerte Celular , Proteínas del Sistema Complemento/metabolismo , Humanos , Ratones , Viabilidad Microbiana , Oxidación-Reducción , Dominios Proteicos , Proteoma/metabolismo , Proteómica , Infecciones por Pseudomonas/sangre , Especificidad por Sustrato , Transcripción Genética , Virulencia , Factores de Virulencia/metabolismo
12.
Plant Mol Biol ; 105(6): 601-610, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33527202

RESUMEN

KEY MESSAGE: We developed two CNNs for predicting ubiquitination sites in Arabidopsis thaliana, demonstrated their competitive performance, analyzed amino acid physicochemical properties and the CNN structures, and predicted ubiquitination sites in Arabidopsis. As an important posttranslational protein modification, ubiquitination plays critical roles in plant physiology, including plant growth and development, biotic and abiotic stress, metabolism, and so on. A lot of ubiquitination site prediction models have been developed for human, mouse and yeast. However, there are few models to predict ubiquitination sites for the plant Arabidopsis thaliana. Based on this context, we proposed two convolutional neural network (CNN) based models for predicting ubiquitination sites in A. thaliana. The two models reach AUC (area under the ROC curve) values of 0.924 and 0.913 respectively in five-fold cross-validation, and 0.921 and 0.914 respectively in independent test, which outperform other models and demonstrate the competitive edge of them. We in-depth analyze the amino acid physicochemical properties in the neighboring sequence regions of the ubiquitination sites, and study the influence of the CNN structure to the prediction performance. Potential ubiquitination sites in the global Arbidopsis proteome are predicted using the two CNN models. To facilitate the community, the source code, training and test dataset, predicted ubiquitination sites in the Arbidopsis proteome are available at GitHub ( http://github.com/nongdaxiaofeng/CNNAthUbi ) for interest users.


Asunto(s)
Arabidopsis/metabolismo , Biología Computacional/métodos , Redes Neurales de la Computación , Ubiquitinación , Aminoácidos/metabolismo , Animales , Humanos , Ratones , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Programas Informáticos , Levaduras
13.
Nat Commun ; 12(1): 891, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33563959

RESUMEN

Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.


Asunto(s)
Metilhistidinas/metabolismo , Metiltransferasas/metabolismo , Proteoma/metabolismo , Secuencias de Aminoácidos , Animales , Células Cultivadas , Histidina/metabolismo , Humanos , Mamíferos/clasificación , Mamíferos/genética , Mamíferos/metabolismo , Metilación , Metiltransferasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mutación , Procesamiento Proteico-Postraduccional , Proteoma/química , Especificidad por Sustrato , Zinc/metabolismo
14.
Nat Commun ; 12(1): 1020, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589611

RESUMEN

The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N,N-dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation.


Asunto(s)
Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Páncreas/metabolismo , Proteoma/genética , Adolescente , Adulto , Niño , Preescolar , Cromatografía Liquida , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/clasificación , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Feto , Técnica del Anticuerpo Fluorescente , Ontología de Genes , Humanos , Masculino , Persona de Mediana Edad , Anotación de Secuencia Molecular , Organogénesis/genética , Páncreas/crecimiento & desarrollo , Proteoma/clasificación , Proteoma/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem
15.
Nature ; 590(7847): 649-654, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33627808

RESUMEN

The cell cycle, over which cells grow and divide, is a fundamental process of life. Its dysregulation has devastating consequences, including cancer1-3. The cell cycle is driven by precise regulation of proteins in time and space, which creates variability between individual proliferating cells. To our knowledge, no systematic investigations of such cell-to-cell proteomic variability exist. Here we present a comprehensive, spatiotemporal map of human proteomic heterogeneity by integrating proteomics at subcellular resolution with single-cell transcriptomics and precise temporal measurements of individual cells in the cell cycle. We show that around one-fifth of the human proteome displays cell-to-cell variability, identify hundreds of proteins with previously unknown associations with mitosis and the cell cycle, and provide evidence that several of these proteins have oncogenic functions. Our results show that cell cycle progression explains less than half of all cell-to-cell variability, and that most cycling proteins are regulated post-translationally, rather than by transcriptomic cycling. These proteins are disproportionately phosphorylated by kinases that regulate cell fate, whereas non-cycling proteins that vary between cells are more likely to be modified by kinases that regulate metabolism. This spatially resolved proteomic map of the cell cycle is integrated into the Human Protein Atlas and will serve as a resource for accelerating molecular studies of the human cell cycle and cell proliferation.


Asunto(s)
Ciclo Celular , Proteogenómica/métodos , Análisis de la Célula Individual/métodos , Transcriptoma , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Linaje de la Célula , Proliferación Celular , Humanos , Interfase , Mitosis , Proteínas Oncogénicas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Proteoma/metabolismo , Factores de Tiempo
16.
Nat Commun ; 12(1): 1279, 2021 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-33627659

RESUMEN

Blood circulating proteins are confounded readouts of the biological processes that occur in different tissues and organs. Many proteins have been linked to complex disorders and are also under substantial genetic control. Here, we investigate the associations between over 1000 blood circulating proteins and body mass index (BMI) in three studies including over 4600 participants. We show that BMI is associated with widespread changes in the plasma proteome. We observe 152 replicated protein associations with BMI. 24 proteins also associate with a genome-wide polygenic score (GPS) for BMI. These proteins are involved in lipid metabolism and inflammatory pathways impacting clinically relevant pathways of adiposity. Mendelian randomization suggests a bi-directional causal relationship of BMI with LEPR/LEP, IGFBP1, and WFIKKN2, a protein-to-BMI relationship for AGER, DPT, and CTSA, and a BMI-to-protein relationship for another 21 proteins. Combined with animal model and tissue-specific gene expression data, our findings suggest potential therapeutic targets further elucidating the role of these proteins in obesity associated pathologies.


Asunto(s)
Obesidad/metabolismo , Proteoma/metabolismo , Adulto , Anciano , Índice de Masa Corporal , Femenino , Humanos , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Masculino , Análisis de la Aleatorización Mendeliana , Persona de Mediana Edad , Obesidad/genética , Proteómica/métodos
17.
Nat Commun ; 12(1): 1269, 2021 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-33627664

RESUMEN

Telomere maintenance by telomerase activation or alternative lengthening of telomeres (ALT) is a major determinant of poor outcome in neuroblastoma. Here, we screen for ALT in primary and relapsed neuroblastomas (n = 760) and characterize its features using multi-omics profiling. ALT-positive tumors are molecularly distinct from other neuroblastoma subtypes and enriched in a population-based clinical sequencing study cohort for relapsed cases. They display reduced ATRX/DAXX complex abundance, due to either ATRX mutations (55%) or low protein expression. The heterochromatic histone mark H3K9me3 recognized by ATRX is enriched at the telomeres of ALT-positive tumors. Notably, we find a high frequency of telomeric repeat loci with a neuroblastoma ALT-specific hotspot on chr1q42.2 and loss of the adjacent chromosomal segment forming a neo-telomere. ALT-positive neuroblastomas proliferate slowly, which is reflected by a protracted clinical course of disease. Nevertheless, children with an ALT-positive neuroblastoma have dismal outcome.


Asunto(s)
Secuenciación Completa del Genoma/métodos , Western Blotting , Exones/genética , Citometría de Flujo , Humanos , Proteoma/metabolismo , Estudios Retrospectivos , Análisis de Secuencia de ARN/métodos , Telómero/genética , Telómero/metabolismo , Homeostasis del Telómero/genética , Proteína Nuclear Ligada al Cromosoma X/genética
18.
Nat Commun ; 12(1): 858, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558502

RESUMEN

Synaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by 'crowding' in the vesicle membrane from stable interaction modules.


Asunto(s)
Reactivos de Enlaces Cruzados/química , Espectrometría de Masas , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Encéfalo/metabolismo , Fusión de Membrana , Unión Proteica , Mapas de Interacción de Proteínas , Proteolípidos , Proteoma/metabolismo , Ratas , Membranas Sinápticas/ultraestructura , Vesículas Sinápticas/ultraestructura , Sinaptofisina/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo
19.
Mol Immunol ; 131: 1-5, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33440289

RESUMEN

Helminths can interact with their hosts in many different ways, including through the secretion of soluble molecules (such as lipids, glycans and proteins) and extracellular vesicles (EVs). The field of helminth secreted EVs has significantly advanced in recent years, mainly due to the molecular characterisation of EV proteomes and research highlighting the potential of EVs and their constituent molecules in the diagnosis and control of parasitic infections. Despite these advancements, the lack of appropriate isolation and purification methods is impeding the discovery of suitable biomarkers for the differentiation of helminth EV populations. In the present review we offer our viewpoint on the different proteomic techniques and approaches that have been developed, as well as solutions to common pitfalls and challenges that could be applied to advance the study of helminth EVs.


Asunto(s)
Vesículas Extracelulares/metabolismo , Helmintos/metabolismo , Proteoma/metabolismo , Animales , Biomarcadores/metabolismo , Helmintos/patogenicidad , Enfermedades Parasitarias/parasitología , Proteómica/métodos
20.
Nat Struct Mol Biol ; 28(2): 143-151, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33432246

RESUMEN

The prevalent model for cataract formation in the eye lens posits that damaged crystallin proteins form light-scattering aggregates. The α-crystallins are thought to counteract this process as chaperones by sequestering misfolded crystallin proteins. In this scenario, chaperone pool depletion would result in lens opacification. Here we analyze lenses from different mouse strains that develop early-onset cataract due to point mutations in α-, ß-, or γ-crystallin proteins. We find that these mutant crystallins are unstable in vitro; in the lens, their levels are substantially reduced, and they do not accumulate in the water-insoluble fraction. Instead, all the other crystallin proteins, including the α-crystallins, are found to precipitate. The changes in protein composition and spatial organization of the crystallins observed in the mutant lenses suggest that the imbalance in the lenticular proteome and altered crystallin interactions are the bases for cataract formation, rather than the aggregation propensity of the mutant crystallins.


Asunto(s)
Catarata/metabolismo , Cristalinas/metabolismo , Cristalino , Agregación Patológica de Proteínas , Animales , Cristalino/metabolismo , Cristalino/patología , Ratones , Chaperonas Moleculares/metabolismo , Proteoma/metabolismo
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