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1.
Cell ; 184(15): 4073-4089.e17, 2021 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-34214469

RESUMEN

Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.


Asunto(s)
Especificidad de Órganos , Mapeo de Interacción de Proteínas , Animales , Marcaje Isotópico , Masculino , Mamíferos , Ratones Endogámicos C57BL , Reproducibilidad de los Resultados
2.
J Virol ; 96(17): e0069922, 2022 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-35993738

RESUMEN

Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of plus-stranded RNA viruses. VPg is covalently linked to the 5' end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses have multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use cricket paralysis virus (CrPV), a dicistrovirus that has four nonidentical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses contain two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1 to VPg4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, mutations of which in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant negative effect. Using viral minigenome reporters that monitor dicistrovirus 5' untranslated (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of distinct IRES translation activities. We uncovered a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote infection. IMPORTANCE Genetic duplication is exceedingly rare in small RNA viral genomes, as there is selective pressure to prevent RNA genomes from expanding. However, some small RNA viruses encode multiple copies of a viral protein, most notably an unusual viral protein that is linked to the viral RNA genome. Here, we investigate a family of viruses that contains multiple viral protein genome-linked proteins and reveal a novel viral strategy whereby viral protein copy number counterbalances differences in viral protein synthesis mechanisms.


Asunto(s)
Dicistroviridae , Genoma Viral , Biosíntesis de Proteínas , Infecciones por Virus ARN , ARN Viral , Proteínas Virales , Regiones no Traducidas 5'/genética , Animales , Línea Celular , Dicistroviridae/genética , Dicistroviridae/metabolismo , Drosophila/citología , Drosophila/virología , Genoma Viral/genética , Sitios Internos de Entrada al Ribosoma/genética , Mutación , Infecciones por Virus ARN/virología , ARN Viral/genética , Serina/metabolismo , Treonina/metabolismo , Carga Viral , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Proteínas Virales/metabolismo
3.
Nucleic Acids Res ; 48(12): 6855-6873, 2020 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-32406909

RESUMEN

Cells limit energy-consuming mRNA translation during stress to maintain metabolic homeostasis. Sequestration of mRNAs by RNA binding proteins (RBPs) into RNA granules reduces their translation, but it remains unclear whether RBPs also function in partitioning of specific transcripts to polysomes (PSs) to guide selective translation and stress adaptation in cancer. To study transcript partitioning under cell stress, we catalogued mRNAs enriched in prostate carcinoma PC-3 cell PSs, as defined by polysome fractionation and RNA sequencing (RNAseq), and compared them to mRNAs complexed with the known SG-nucleator protein, G3BP1, as defined by spatially-restricted enzymatic tagging and RNAseq. By comparing these compartments before and after short-term arsenite-induced oxidative stress, we identified three major categories of transcripts, namely those that were G3BP1-associated and PS-depleted, G3BP1-dissociated and PS-enriched, and G3BP1-associated but also PS-enriched. Oxidative stress profoundly altered the partitioning of transcripts between these compartments. Under arsenite stress, G3BP1-associated and PS-depleted transcripts correlated with reduced expression of encoded mitochondrial proteins, PS-enriched transcripts that disassociated from G3BP1 encoded cell cycle and cytoprotective proteins whose expression increased, while transcripts that were both G3BP1-associated and PS-enriched encoded proteins involved in diverse stress response pathways. Therefore, G3BP1 guides transcript partitioning to reprogram mRNA translation and support stress adaptation.


Asunto(s)
ADN Helicasas/genética , Estrés Oxidativo/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Biosíntesis de Proteínas/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , ARN Mensajero/genética , Arsenitos/toxicidad , Carcinoma/genética , Carcinoma/metabolismo , Gránulos Citoplasmáticos/genética , Metabolismo Energético/genética , Humanos , Masculino , Estrés Oxidativo/efectos de los fármacos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas de Unión al ARN/genética
4.
BMC Genomics ; 16: 63, 2015 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-25757461

RESUMEN

BACKGROUND: The Western honey bee (Apis mellifera L.) is a critical component of human agriculture through its pollination activities. For years, beekeepers have controlled deadly pathogens such as Paenibacillus larvae, Nosema spp. and Varroa destructor with antibiotics and pesticides but widespread chemical resistance is appearing and most beekeepers would prefer to eliminate or reduce the use of in-hive chemicals. While such treatments are likely to still be needed, an alternate management strategy is to identify and select bees with heritable traits that allow them to resist mites and diseases. Breeding such bees is difficult as the tests involved to identify disease-resistance are complicated, time-consuming, expensive and can misidentify desirable genotypes. Additionally, we do not yet fully understand the mechanisms behind social immunity. Here we have set out to discover the molecular mechanism behind hygienic behavior (HB), a trait known to confer disease resistance in bees. RESULTS: After confirming that HB could be selectively bred for, we correlated measurements of this behavior with protein expression over a period of three years, at two geographically distinct sites, using several hundred bee colonies. By correlating the expression patterns of individual proteins with HB scores, we identified seven putative biomarkers of HB that survived stringent control for multiple hypothesis testing. Intriguingly, these proteins were all involved in semiochemical sensing (odorant binding proteins), nerve signal transmission or signal decay, indicative of the series of events required to respond to an olfactory signal from dead or diseased larvae. We then used recombinant versions of two odorant-binding proteins to identify the classes of ligands that these proteins might be helping bees detect. CONCLUSIONS: Our data suggest that neurosensory detection of odors emitted by dead or diseased larvae is the likely mechanism behind a complex and important social immunity behavior that allows bees to co-exist with pathogens.


Asunto(s)
Abejas/genética , Conducta Animal/fisiología , Resistencia a la Enfermedad/genética , Sistemas Neurosecretores , Agricultura , Animales , Abejas/parasitología , Genotipo , Humanos , Larva , Nosema/patogenicidad , Odorantes , Polinización/genética , Transducción de Señal/genética , Varroidae/genética , Varroidae/patogenicidad
5.
BMC Microbiol ; 15: 206, 2015 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-26453029

RESUMEN

BACKGROUND: The pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in individuals suffering from HIV/AIDS. The cyclic-AMP/protein kinase A (PKA) signal transduction pathway regulates the production of extracellular virulence factors in C. neoformans, but the influence of the pathway on the secretome has not been investigated. In this study, we performed quantitative proteomics using galactose-inducible and glucose-repressible expression of the PKA1 gene encoding the catalytic subunit of PKA to identify regulated proteins in the secretome. METHODS: The proteins in the supernatants of cultures of C. neoformans were precipitated and identified using liquid chromatography-coupled tandem mass spectrometry. We also employed multiple reaction monitoring in a targeted approach to identify fungal proteins in samples from macrophages after phagocytosis of C. neoformans cells, as well as from the blood and bronchoalveolar fluid of infected mice. RESULTS: We identified 61 secreted proteins and found that changes in PKA1 expression influenced the extracellular abundance of five proteins, including the Cig1 and Aph1 proteins with known roles in virulence. We also observed a change in the secretome profile upon induction of Pka1 from proteins primarily involved in catabolic and metabolic processes to an expanded set that included proteins for translational regulation and the response to stress. We further characterized the secretome data using enrichment analysis and by predicting conventional versus non-conventional secretion. Targeted proteomics of the Pka1-regulated proteins allowed us to identify the secreted proteins in lysates of phagocytic cells containing C. neoformans, and in samples from infected mice. This analysis also revealed that modulation of PKA1 expression influences the intracellular survival of cryptococcal cells upon phagocytosis. CONCLUSIONS: Overall, we found that the cAMP/PKA pathway regulates specific components of the secretome including proteins that affect the virulence of C. neoformans. The detection of secreted cryptococcal proteins from infected phagocytic cells and tissue samples suggests their potential utility as biomarkers of infection. The proteomics data are available via ProteomeXchange with identifiers PXD002731 and PASS00736.


Asunto(s)
Biomarcadores/análisis , Cryptococcus neoformans/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Proteoma/análisis , Factores de Virulencia/metabolismo , Animales , Precipitación Química , Cromatografía Liquida , Medios de Cultivo/química , AMP Cíclico/metabolismo , Femenino , Macrófagos/microbiología , Ratones Endogámicos BALB C , Espectrometría de Masas en Tándem
6.
Mol Cell Proteomics ; 11(9): 692-709, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22661456

RESUMEN

Type III secretion systems are central to the pathogenesis and virulence of many important Gram-negative bacterial pathogens, and elucidation of the secretion mechanism and identification of the secreted substrates are critical to our understanding of their pathogenic mechanisms and developing potential therapeutics. Stable isotope labeling with amino acids in cell culture-based mass spectrometry is a quantitative and highly sensitive proteomics tool that we have previously used to successfully analyze the type III secretomes of Citrobacter rodentium and Salmonella enterica serovar Typhimurium. In this report, stable isotope labeling with amino acids in cell culture was used to analyze the type III secretome of enteropathogenic Escherichia coli (EPEC), an important human pathogen, which, together with enterohemorrhagic E. coli and C. rodentium, represents the family of attaching and effacing bacterial pathogens. We not only confirmed all 25 known EPEC type III-secreted proteins and effectors previously identified by conventional molecular and bioinformatical techniques but also identified several new type III-secreted proteins, including two novel effectors, C_0814/NleJ and LifA, that were shown to be translocated into host cells. LifA is a known virulence factor believed to act as a toxin as well as an adhesin, but its mechanism of secretion and function is not understood. With a predicted molecular mass of 366 kDa, LifA is the largest type III effector identified thus far in any pathogen. We further demonstrated that Efa1, ToxB, and Z4332 (homologs of LifA in enterohemorrhagic E. coli) are also type III effectors. This study has comprehensively characterized the type III secretome of EPEC, expanded the repertoire of type III-secreted effectors for the attaching and effacing pathogens, and provided new insights into the mode of function for LifA/Efa1/ToxB/Z4332, an important family of virulence factors.


Asunto(s)
Sistemas de Secreción Bacterianos , Escherichia coli Enteropatógena/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/análisis , Secuencia de Aminoácidos , Adhesión Bacteriana , Toxinas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/metabolismo , Escherichia coli Enteropatógena/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Marcaje Isotópico , Proteoma
7.
J Biol Chem ; 287(39): 32324-37, 2012 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-22810234

RESUMEN

The co-evolutionary relationship between pathogen and host has led to a regulatory cycle between virulence factors needed for survival and antivirulence factors required for host transmission. This is exemplified in Salmonella spp. by the zirTS antivirulence genes: a secretion pathway comprised of the outer membrane transporter ZirT, and its secreted partner, ZirS. ZirTS act within the gastrointestinal tract to function as a virulence modulator and during Salmonella shedding in anticipation of a new host. Together, ZirT and ZirS decrease virulence by lowering bacterial colonization at systemic sites through an unknown mechanism. To understand this mechanism, we have probed the zirTS pathway both structurally and biochemically. The NMR derived structural ensemble of the C-terminal domain of ZirS reveals an immunoglobin superfamily fold (IgSF). Stable isotope labeling by amino acids in cell culture experiments show that the ZirS IgSF domain interacts with its transporter ZirT, and reveal a new protein interaction partner of the pathway, a protein encoded adjacent to zirTS that we have designated as ZirU. ZirU is secreted by ZirT and is also a predicted IgSF. Biochemical analysis delineates ZirT into an N-terminal porin-like ß domain and C-terminal extracellular soluble IgSF domain, whereas biophysical characterization suggests that the transporter undergoes self-association in a concentration-dependent manner. We observe that ZirS and ZirU directly interact with each other and with the extracellular domains of ZirT. Here we show that the zir antivirulence pathway is a multiprotein immunoglobulin adhesion system consisting of a complex interplay between ZirS, ZirT, and ZirU.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Complejos Multiproteicos/química , Salmonella typhimurium/química , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/fisiología , Proteínas Portadoras/genética , Espectroscopía de Resonancia Magnética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad
8.
Folia Med (Plovdiv) ; 65(3): 490-494, 2023 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-38351827

RESUMEN

The epiphrenic esophageal diverticulum is a rare non-malignant condition that is commonly associated with motility disorders. It would normally be treated surgically, but with the advancement of endoscopy techniques, peroral endoscopic myotomy with septotomy (D-POEM) has shown its benefits in coping with the symptoms. We present a case of a 71-year-old woman with increasing symptoms of dysphagia, weight loss and imaging data showing a large epiphrenic diverticulum. We treated her using peroral endoscopic myotomy combined with septotomy of the diverticular septum. The procedure showed excellent results with reducing the amount of contrast materials retained in it, improving the quality of life of the patient, and increasing her weight. There were minimal adverse events and no perforations or severe adverse effects occurred. D-POEM is a new and rapidly evolving procedure that is proving to be a safe and effective method of treating epiphrenic esophageal diverticulum.


Asunto(s)
Procedimientos Quirúrgicos del Sistema Digestivo , Divertículo Esofágico , Miotomía , Humanos , Femenino , Anciano , Calidad de Vida , Miotomía/efectos adversos , Miotomía/métodos , Divertículo Esofágico/cirugía , Divertículo Esofágico/diagnóstico , Divertículo Esofágico/etiología , Esófago , Resultado del Tratamiento
9.
J Biol Chem ; 286(27): 24023-35, 2011 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-21566117

RESUMEN

Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.


Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/fisiología , Islas Genómicas/fisiología , Interacciones Huésped-Patógeno/fisiología , Proteínas de la Membrana/metabolismo , Salmonella typhimurium/fisiología , Salmonella typhimurium/patogenicidad , Proteínas Bacterianas/genética , Humanos , Proteínas de la Membrana/genética
10.
Folia Med (Plovdiv) ; 64(1): 143-147, 2022 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-35851900

RESUMEN

Endoscopic self-expandable metallic stent (SEMS) decompression in patients with bowel obstruction due to colon carcinoma has been practiced for over two decades now, both in potentially curable cases and metastatic cancer. Using this case series, we aim to review the literature on the subject and to present our initial experience with using this technique as a bridge to single stage surgery, thus minimizing colostomy creation.


Asunto(s)
Neoplasias Colorrectales , Obstrucción Intestinal , Stents Metálicos Autoexpandibles , Neoplasias Colorrectales/complicaciones , Neoplasias Colorrectales/cirugía , Descompresión Quirúrgica/métodos , Humanos , Obstrucción Intestinal/etiología , Obstrucción Intestinal/cirugía , Estudios Retrospectivos , Resultado del Tratamiento
11.
Cell Rep ; 40(3): 111096, 2022 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-35858568

RESUMEN

Accurate and efficient folding of nascent protein sequences into their native states requires support from the protein homeostasis network. Herein we probe which newly translated proteins are thermo-sensitive, making them susceptible to misfolding and aggregation under heat stress using pulse-SILAC mass spectrometry. We find a distinct group of proteins that is highly sensitive to this perturbation when newly synthesized but not once matured. These proteins are abundant and highly structured. Notably, they display a tendency to form ß sheet secondary structures, have more complex folding topology, and are enriched for chaperone-binding motifs, suggesting a higher demand for chaperone-assisted folding. These polypeptides are also more often components of stable protein complexes in comparison with other proteins. Combining these findings suggests the existence of a specific subset of proteins in the cell that is particularly vulnerable to misfolding and aggregation following synthesis before reaching the native state.


Asunto(s)
Pliegue de Proteína , Proteoma , Chaperonas Moleculares/metabolismo , Péptidos/metabolismo , Unión Proteica , Proteoma/metabolismo
12.
J Proteome Res ; 10(3): 1139-50, 2011 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-21166474

RESUMEN

Mortality attributable to infection with methicillin-resistant Staphylococcus aureus (MRSA) has now overtaken the death rate for AIDS in the United States, and advances in research are urgently needed to address this challenge. We report the results of the systematic identification of protein-protein interactions for the hospital-acquired strain MRSA-252. Using a high-throughput pull-down strategy combined with quantitative proteomics to distinguish specific from nonspecific interactors, we identified 13,219 interactions involving 608 MRSA proteins. Consecutive analyses revealed that this protein interaction network (PIN) exhibits scale-free organization with the characteristic presence of highly connected hub proteins. When clinical and experimental antimicrobial targets were queried in the network, they were generally found to occupy peripheral positions in the PIN with relatively few interacting partners. In contrast, the hub proteins identified in this MRSA PIN that are essential for network integrity and stability have largely been overlooked as drug targets. Thus, this empirical MRSA-252 PIN provides a rich source for identifying critical proteins essential for network stability, many of which can be considered as prospective antimicrobial drug targets.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/metabolismo , Mapeo de Interacción de Proteínas/métodos , Animales , Proteínas Bacterianas/genética , Humanos , Espectrometría de Masas , Proteómica/métodos , Proteínas Recombinantes de Fusión/metabolismo , Infecciones Estafilocócicas/metabolismo
13.
Genome Biol ; 21(1): 140, 2020 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-32539747

RESUMEN

BACKGROUND: The type I interferon (IFN) response is an ancient pathway that protects cells against viral pathogens by inducing the transcription of hundreds of IFN-stimulated genes. Comprehensive catalogs of IFN-stimulated genes have been established across species and cell types by transcriptomic and biochemical approaches, but their antiviral mechanisms remain incompletely characterized. Here, we apply a combination of quantitative proteomic approaches to describe the effects of IFN signaling on the human proteome, and apply protein correlation profiling to map IFN-induced rearrangements in the human protein-protein interaction network. RESULTS: We identify > 26,000 protein interactions in IFN-stimulated and unstimulated cells, many of which involve proteins associated with human disease and are observed exclusively within the IFN-stimulated network. Differential network analysis reveals interaction rewiring across a surprisingly broad spectrum of cellular pathways in the antiviral response. We identify IFN-dependent protein-protein interactions mediating novel regulatory mechanisms at the transcriptional and translational levels, with one such interaction modulating the transcriptional activity of STAT1. Moreover, we reveal IFN-dependent changes in ribosomal composition that act to buffer IFN-stimulated gene protein synthesis. CONCLUSIONS: Our map of the IFN interactome provides a global view of the complex cellular networks activated during the antiviral response, placing IFN-stimulated genes in a functional context, and serves as a framework to understand how these networks are dysregulated in autoimmune or inflammatory disease.


Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Interferón Tipo I/metabolismo , Mapas de Interacción de Proteínas , Proteoma , Virosis/metabolismo , Humanos , Proteómica , Proteínas Ribosómicas/metabolismo , Transducción de Señal
14.
mSystems ; 5(3)2020 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-32487743

RESUMEN

Enteropathogenic Escherichia coli (EPEC) causes severe diarrheal disease and is present globally. EPEC virulence requires a bacterial type III secretion system to inject >20 effector proteins into human intestinal cells. Three effectors travel to mitochondria and modulate apoptosis; however, the mechanisms by which effectors control apoptosis from within mitochondria are unknown. To identify and quantify global changes in mitochondrial proteolysis during infection, we applied the mitochondrial terminal proteomics technique mitochondrial stable isotope labeling by amino acids in cell culture-terminal amine isotopic labeling of substrates (MS-TAILS). MS-TAILS identified 1,695 amino N-terminal peptides from 1,060 unique proteins and 390 N-terminal peptides from 215 mitochondrial proteins at a false discovery rate of 0.01. Infection modified 230 cellular and 40 mitochondrial proteins, generating 27 cleaved mitochondrial neo-N termini, demonstrating altered proteolytic processing within mitochondria. To distinguish proteolytic events specific to EPEC from those of canonical apoptosis, we compared mitochondrial changes during infection with those reported from chemically induced apoptosis. During infection, fewer than half of all mitochondrial cleavages were previously described for canonical apoptosis, and we identified nine mitochondrial proteolytic sites not previously reported, including several in proteins with an annotated role in apoptosis, although none occurred at canonical Asp-Glu-Val-Asp (DEVD) sites associated with caspase cleavage. The identification and quantification of novel neo-N termini evidences the involvement of noncaspase human or EPEC protease(s) resulting from mitochondrial-targeting effectors that modulate cell death upon infection. All proteomics data are available via ProteomeXchange with identifier PXD016994IMPORTANCE To our knowledge, this is the first study of the mitochondrial proteome or N-terminome during bacterial infection. Identified cleavage sites that had not been previously reported in the mitochondrial N-terminome and that were not generated in canonical apoptosis revealed a pathogen-specific strategy to control human cell apoptosis. These data inform new mechanisms of virulence factors targeting mitochondria and apoptosis during infection and highlight how enteropathogenic Escherichia coli (EPEC) manipulates human cell death pathways during infection, including candidate substrates of an EPEC protease within mitochondria. This understanding informs the development of new antivirulence strategies against the many human pathogens that target mitochondria during infection. Therefore, mitochondrial stable isotope labeling by amino acids in cell culture-terminal amine isotopic labeling of substrates (MS-TAILS) is useful for studying other pathogens targeting human cell compartments.

15.
Front Neurosci ; 13: 143, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30941001

RESUMEN

Extracellular matrix (ECM) remodeling, degradation and glioma cell motility are critical aspects of glioblastoma multiforme (GBM). Despite being a rich source of potential biomarkers and targets for therapeutic advance, the dynamic changes occurring within the extracellular environment that are specific to GBM motility have yet to be fully resolved. The gap junction protein connexin43 (Cx43) increases glioma migration and invasion in a variety of in vitro and in vivo models. In this study, the upregulation of Cx43 in C6 glioma cells induced morphological changes and the secretion of proteins associated with cell motility. Demonstrating the selective engagement of ECM remodeling networks, secretome analysis revealed the near-binary increase of osteopontin and matrix metalloproteinase-3 (MMP3), with gelatinase and NFF-3 assays confirming the proteolytic activities. Informatic analysis of interactome and secretome downstream of Cx43 identifies networks of glioma motility that appear to be synergistically engaged. The data presented here implicate ECM remodeling and matrikine signals downstream of Cx43/MMP3/osteopontin and ARK1B10 inhibition as possible avenues to inhibit GBM.

16.
Sci Rep ; 7(1): 8442, 2017 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-28814753

RESUMEN

The yeast Sup35 protein is a subunit of the translation termination factor, and its conversion to the [PSI +] prion state leads to more translational read-through. Although extensive studies have been done on [PSI +], changes at the proteomic level have not been performed exhaustively. We therefore used a SILAC-based quantitative mass spectrometry approach and identified 4187 proteins from both [psi -] and [PSI +] strains. Surprisingly, there was very little difference between the two proteomes under standard growth conditions. We found however that several [PSI +] strains harbored an additional chromosome, such as chromosome I. Albeit, we found no evidence to support that [PSI +] induces chromosomal instability (CIN). Instead we hypothesized that the selective pressure applied during the establishment of [PSI +]-containing strains could lead to a supernumerary chromosome due to the presence of the ade1-14 selective marker for translational read-through. We therefore verified that there was no prevalence of disomy among newly generated [PSI +] strains in absence of strong selection pressure. We also noticed that low amounts of adenine in media could lead to higher levels of mitochondrial DNA in [PSI +] in ade1-14 cells. Our study has important significance for the establishment and manipulation of yeast strains with the Sup35 prion.


Asunto(s)
Aneuploidia , Factores de Terminación de Péptidos/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Inestabilidad Cromosómica/genética , Cromosomas Fúngicos/genética , ADN de Hongos/química , ADN de Hongos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Espectrometría de Masas/métodos , Factores de Terminación de Péptidos/genética , Proteoma/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
17.
J Proteomics ; 77: e1-10, 2012 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-22967496

RESUMEN

Messenger RNA-binding translational regulatory proteins determine in large part the spectrum of transcripts that are translated under specific cellular contexts. Y-box binding protein-1 (YB-1) is a conserved eukaryotic translational regulator that is implicated in cancer progression. To identify specific proteins that are translationally regulated by YB-1, we established a pulse-labelling approach combining Click chemistry and stable isotope labelling by amino acids in cell culture (SILAC). The proteome of TC32 human Ewing sarcoma cells, which robustly express YB-1, was compared with or without YB-1 siRNA knockdown. Cells labelled with light or heavy isotopologs of Arg and Lys were then cotranslationally pulsed with the methionine derivative, azidohomoalanine (AHA). Cells were lysed and newly synthesized proteins were selectively derivatized via a Click (3+2 cycloaddition) reaction to add an alkyne biotin tag. They were then affinity purified and subjected to liquid chromatography-tandem mass spectrometry. This combined Click-SILAC approach enabled us to catalog and quantify newly synthesized proteins regulated by YB-1 after only 45 min of labelling. Bioinformatic analysis revealed that YB-1 regulated proteins are involved in diverse biological pathways. We anticipate that this Click-SILAC strategy will be useful for studying short-term protein synthesis in different cell culture systems and under diverse biological contexts.


Asunto(s)
Neoplasias Óseas/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Biosíntesis de Proteínas , Proteoma/biosíntesis , Sarcoma de Ewing/metabolismo , Proteína 1 de Unión a la Caja Y/biosíntesis , Neoplasias Óseas/patología , Línea Celular Tumoral , Humanos , Marcaje Isotópico , Proteómica/métodos , Sarcoma de Ewing/patología
18.
ACS Chem Biol ; 7(2): 350-9, 2012 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-22066782

RESUMEN

We have recently mapped the protein interaction network of methicillin-resistant Staphylococcus aureus (MRSA), which revealed its scale-free organization with characteristic presence of highly connected hub proteins that are critical for bacterial survival. Here we report the discovery of inhibitors that are highly potent against one such hub target, staphylococcal pyruvate kinase (PK). Importantly, the developed compounds demonstrate complete selectivity for the bacterial enzyme compared to all human orthologues. The lead 91nM inhibitor IS-130 has been identified through ligand-based cheminformatic exploration of a chemical space around micromolar hits initially generated by experimental screening. The following crystallographic study resulted in identification of a tetrameric MRSA PK structure where IS-130 is bound to the interface between the protein's subunits. This newly described binding pocket is not present in otherwise highly similar human orthologues and can be effectively utilized for selective inhibition of bacterial PK. The following synthetic modifications of IS-130, guided by structure-based molecular modeling, resulted in the development of MRSA PK inhibitors with much improved antimicrobial properties. Considering a notable lack of recent reports on novel antibacterial targets and cognate antibacterial compounds, this study provides a valuable perspective on the development of a new generation of antimicrobials. Equally noteworthy, the results of the current work highlight the importance of rigorous cheminformatics-based exploration of the results of high-throughput experiments.


Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/enzimología , Piruvato Quinasa/antagonistas & inhibidores , Piruvato Quinasa/metabolismo , Secuencia de Aminoácidos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mapas de Interacción de Proteínas/efectos de los fármacos , Piruvato Quinasa/química , Alineación de Secuencia , Infecciones Estafilocócicas/tratamiento farmacológico
19.
PLoS One ; 6(4): e19247, 2011 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-21559420

RESUMEN

One of the major challenges in management of spinal cord injury (SCI) is that the assessment of injury severity is often imprecise. Identification of reliable, easily quantifiable biomarkers that delineate the severity of the initial injury and that have prognostic value for the degree of functional recovery would significantly aid the clinician in the choice of potential treatments. To find such biomarkers we performed quantitative liquid chromatography-mass spectrometry (LC-MS/MS) analyses of cerebrospinal fluid (CSF) collected from rats 24 h after either a moderate or severe SCI. We identified a panel of 42 putative biomarkers of SCI, 10 of which represent potential biomarkers of SCI severity. Three of the candidate biomarkers, Ywhaz, Itih4, and Gpx3 were also validated by Western blot in a biological replicate of the injury. The putative biomarkers identified in this study may potentially be a valuable tool in the assessment of the extent of spinal cord damage.


Asunto(s)
Biomarcadores/líquido cefalorraquídeo , Traumatismos de la Médula Espinal/líquido cefalorraquídeo , Traumatismos de la Médula Espinal/diagnóstico , Animales , Biomarcadores/metabolismo , Western Blotting , Cromatografía Liquida/métodos , Masculino , Espectrometría de Masas/métodos , Péptidos/química , Pronóstico , Proteómica/métodos , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Factores de Tiempo
20.
J Immunol ; 180(4): 2459-65, 2008 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-18250455

RESUMEN

Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-A(b)) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.


Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Antígenos de Diferenciación de Linfocitos T/aislamiento & purificación , Chlamydia muridarum/inmunología , Líquido Intracelular/inmunología , Líquido Intracelular/microbiología , Proteoma/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/uso terapéutico , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos de Diferenciación de Linfocitos T/uso terapéutico , Células Cultivadas , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/prevención & control , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Femenino , Antígenos H-2/aislamiento & purificación , Antígenos H-2/metabolismo , Células HeLa , Antígenos de Histocompatibilidad Clase II/aislamiento & purificación , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase II/uso terapéutico , Humanos , Líquido Intracelular/química , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Péptidos/uso terapéutico , Unión Proteica/inmunología , Proteoma/aislamiento & purificación , Proteoma/metabolismo , Espectrometría de Masas en Tándem
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