RESUMEN
Bisphenol A is an extremely high-volume chemical widely used in polycarbonate plastics, the linings of food and beverage tins, and shopping receipts. Canadians are ubiquitously exposed to bisphenol A and research shows that exposure at environmentally relevant doses causes endocrine disruption. Recent risk assessments and exposure estimates by the European Food Safety Authority have guided increased restrictions around the use of bisphenol A and established a lower tolerable daily intake, while the CLARITY-BPA program in the United States identified several adverse effects below this exposure level. Within the context of bisphenol toxicity and international regulation, this paper describes the need for revised bisphenol A risk assessments in Canada. Completed in 2008, the most recent bisphenol A risk assessment conducted by Health Canada does not include risks from alternative bisphenols or non-dietary exposure. It also does not account for the additive effects caused by simultaneous exposure to multiple endocrine-disrupting chemicals.
Asunto(s)
Compuestos de Bencidrilo , Disruptores Endocrinos , Compuestos de Bencidrilo/análisis , Compuestos de Bencidrilo/toxicidad , Canadá , Disruptores Endocrinos/toxicidad , Fenoles/análisis , Fenoles/toxicidadRESUMEN
Protein-protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas-mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome-wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.
Asunto(s)
Caspasas/metabolismo , Citoplasma/metabolismo , Mitocondrias/metabolismo , Proteómica/métodos , Receptor fas/metabolismo , Apoptosis , Cromatografía Liquida , Humanos , Marcaje Isotópico , Células Jurkat , Espectrometría de Masas , Mapas de Interacción de Proteínas , ProteolisisRESUMEN
To improve proteome coverage and protein C-terminal identification, we characterized the Methanosarcina acetivorans thermophilic proteinase LysargiNase, which cleaves before lysine and arginine up to 55 °C. Unlike trypsin, LysargiNase-generated peptides had N-terminal lysine or arginine residues and fragmented with b ion-dominated spectra. This improved protein C terminal-peptide identification and several arginine-rich phosphosite assignments. Notably, cleavage also occurred at methylated or dimethylated lysine and arginine, facilitating detection of these epigenetic modifications.
Asunto(s)
Metaloproteasas/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Methanosarcina/enzimología , Metilación , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Especificidad por Sustrato , Tripsina/metabolismoRESUMEN
UNLABELLED: The transcription factors HilA and SsrB activate expression of two type III secretion systems (T3SSs) and cognate effectors that reprogram host cell functions to benefit infecting Salmonella in the host. These transcription factors, the secretion systems, and the effectors are all encoded by horizontally acquired genes. Using quantitative proteomics, we quantified the abundance of 2,149 proteins from hilA or ssrB Salmonella in vitro. Our results suggest that the HilA regulon does not extend significantly beyond proteins known to be involved in direct interactions with intestinal epithelium. On the other hand, SsrB influences the expression of a diverse range of proteins, many of which are ancestral to the acquisition of ssrB. In addition to the known regulon of T3SS-related proteins, we show that, through SodCI and bacterioferritin, SsrB controls resistance to reactive oxygen species and that SsrB down-regulates flagella and motility. This indicates that SsrB-controlled proteins not only redirect host cell membrane traffic to establish a supportive niche within host cells but also have adapted to the chemistry and physical constraints of that niche. IMPORTANCE: Expression of T3SSs typically requires a transcription factor that is linked in a genomic island. Studies of the targets of HilA and SsrB have focused on almost exclusively on T3SS substrates that are either linked or encoded in distinct genomic islands. By broadening our focus, we found that the regulon of SsrB extended considerably beyond T3SS-2 and its substrates, while that of HilA did not. That at least two SsrB-regulated processes streamline existence in the intracellular niche afforded by T3SS-2 seems to be a predictable outcome of evolution and natural selection. However, and importantly, these are the ï¬rst such functions to be implicated as being SsrB dependent. The concept of T3SS-associated transcription factors coordinating manipulations of host cells together with distinct bacterial processes for increased efficiency has unrealized implications for numerous host-pathogen systems.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/genética , Salmonella/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Biología Computacional , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Transferencia de Gen Horizontal , Islas Genómicas , Familia de Multigenes , Fenotipo , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulón , Salmonella/crecimiento & desarrollo , Transactivadores/genética , Factores de Transcripción/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Proteolytic processing is a ubiquitous and irreversible post-translational modification involving limited and highly specific hydrolysis of peptide and isopeptide bonds of a protein by a protease. Cleavage generates shorter protein chains displaying neo-N and -C termini, often with new or modified biological activities. Within the past decade, degradomics and terminomics have emerged as significant proteomics subfields dedicated to characterizing proteolysis products as well as natural protein N and C termini. Here we provide an overview of contemporary proteomics-based methods, including specific quantitation, data analysis, and curation considerations, and highlight exciting new and emerging applications within these fields enabling in vivo analysis of proteolytic events.
Asunto(s)
Técnicas Químicas Combinatorias/métodos , Fragmentos de Péptidos/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Proteómica/métodos , Secuencias de Aminoácidos , Biotinilación , Cromatografía Liquida , Humanos , Marcaje Isotópico , Espectrometría de Masas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Proteolisis , Proteoma/química , Proteómica/instrumentación , Especificidad por SustratoRESUMEN
During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.
Asunto(s)
Proteínas Bacterianas/genética , Islas Genómicas , Proteínas de Choque Térmico HSP27/genética , Macrófagos/metabolismo , Fosfoproteínas/genética , Proteínas Quinasas/genética , Proteoma/genética , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Actinas/genética , Actinas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Línea Celular , Regulación de la Expresión Génica , Proteínas de Choque Térmico HSP27/metabolismo , Células HeLa , Proteínas de Choque Térmico , Interacciones Huésped-Patógeno , Humanos , Macrófagos/citología , Macrófagos/microbiología , Ratones , Chaperonas Moleculares , Fosfoproteínas/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Quinasas/metabolismo , Proteoma/aislamiento & purificación , Proteoma/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Salmonella enterica is a bacterial pathogen that causes gastroenteritis and typhoid fever. Virulence is achieved by two type III secretion systems that translocate effector proteins into host cells, where they mimic or block host protein function. Effectors translocated into host cells by the first type III secretion system facilitate invasion and stimulate intracellular signaling cascades leading to inflammation. Here, we performed global temporal analysis of host signaling events induced during the initial stages of Salmonella infection and identified the dynamics of host protein phosphorylation as well as differences between growth factor-stimulated and bacteria-induced signaling. Informatics analysis predicted that sites with altered phosphorylation in infected cells were targeted by the serine-threonine kinases Akt, protein kinase C, and Pim and that up to half of the host phosphorylation events detected after Salmonella infection required the effector protein SopB. Our data reveal extensive manipulation of host phosphorylation cascades by this Salmonella effector and provide a detailed map of the events leading to intestinal inflammation, which is the crucial host response that enables Salmonella to proliferate in the intestine.
Asunto(s)
Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/fisiología , Proteínas Quinasas/metabolismo , Infecciones por Salmonella/enzimología , Salmonella enterica/fisiología , Transducción de Señal , Células HeLa , Humanos , Fosforilación , Proteómica/métodos , Infecciones por Salmonella/microbiologíaRESUMEN
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/genéticaRESUMEN
Recently, the field of phosphoproteomics has progressed to the point where thousands of protein phosphorylations can be analyzed simultaneously and used to address significant biological questions. However, several challenges still exist in current LC-MS/MS-based phosphoproteomics methods. Among these are the increased dynamic range of phosphoproteomics samples (due to low stoichiometry of most protein phosphorylations), insufficient inhibition of phosphatase activity, and neutral losses which occur during phosphopeptide fragmentation by MS(n). Here we present an improved method, free of conventional phosphatase inhibitors, for sample treatment to minimize phosphatase activity and improve the efficiency of phosphopeptide enrichment. We also present a solution-based IEF method for phosphopeptide fractionation and explore the utility of various fragmentation methods for identifying phosphopeptides and localizing phosphorylation sites.
Asunto(s)
Fraccionamiento Celular/métodos , Cromatografía Liquida/métodos , Fosfopéptidos/análisis , Espectrometría de Masas en Tándem/métodos , Animales , HumanosRESUMEN
Networks of protein-protein and protein-metabolite interactions are commonly found in biological systems where signals must be passed from one location or component within a cell to another, such as from a receptor on the plasma membrane to a transcription factor in the nucleus. Regulation of such networks, or signal transduction pathways, is often achieved by transient, reversible modification of the components involved. Several types of post-translational modifications of proteins are employed in signal transduction including ubiquitylation of lysines and palmitoylation of cysteines, but by far the best appreciated and apparently the most important involves phosphorylation of serine, threonine and tyrosine residues. Whilst protein phosphorylation has long been recognized as functionally important, low stoichiometry has ultimately impeded global analyses (phosphoproteomics). Recent developments in the application of metal oxide chromatography and advanced mass spectrometric techniques have enabled phosphoproteomics to move beyond mere proof-of-principle experiments, to the stage where it can successfully address complex biological questions. Here we cover the development of phosphopeptide/protein analysis by mass spectrometry and the various techniques used to enrich phosphopeptides/proteins. We also speculate on the future of phosphoproteomic research, now that the goal of generating global phosphoproteomic datasets has been realized.
Asunto(s)
Fosfoproteínas/análisis , Proteómica/métodos , Animales , Fraccionamiento Químico , Cromatografía Liquida , Humanos , Espectrometría de Masas , Fosfoproteínas/química , FosforilaciónRESUMEN
Salmonella enterica is a bacterial pathogen responsible for enteritis and typhoid fever. Virulence is linked to two Salmonella pathogenicity islands (SPI-1 and SPI-2) on the bacterial chromosome, each of which encodes a type III secretion system. While both the SPI-1 and SPI-2 systems secrete an array of effectors into the host, relatively few host proteins have been identified as targets for their effects. Here we use stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry-based proteomics to identify the host targets of the SPI-1 effector, SopB/SigD. The only host protein found to bind immunoprecipitated SopB was the small G-protein Cdc42. The interaction was confirmed by reciprocal immunoprecipitation, and Cdc42 also bound glutathione S-transferase-fused SopB and SopB delivered through infection by the bacteria, confirming the interaction by an orthogonal method and in a more physiological context. The region of SopB responsible for the interaction was mapped to residues 117-168, and SopB is ubiquitylated at both K19 and K541, likely as monoubiquitylation. SopB colocalizes with activated Cdc42 near the plasmalemma, but we found no evidence that SopB alone can alter Cdc42 activity. This approach is also widely applicable to identify binding partners to other bacterial effectors.
Asunto(s)
Proteínas Bacterianas/metabolismo , Salmonella enterica/genética , Ubiquitinación , Proteína de Unión al GTP cdc42/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Línea Celular Transformada , Regulación de la Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Eliminación de Secuencia , Proteína de Unión al GTP cdc42/químicaRESUMEN
In metazoans macrophage cells use phagocytosis, the process of engulfing large particles, to control the spread of pathogens in the body, to clear dead or dying cells, and to aid in tissue remodelling, while the same process is also used by unicellular eukaryotes to ingest food. Phagocytosing cells essentially swallow the particles, trapping them in vacuoles called phagosomes that go through a series of maturation steps, culminating in the destruction of the internalized cargo. Because of their central role in innate immunity and their relatively simple structure (one membrane bilayer surrounding a single particle), phagosomes have been a popular subject for organelle proteomics studies. Qualitative proteomic technologies are now very sensitive so hundreds of different proteins have been identified in phagosomes from several species, revealing new properties of these intriguing compartments. More recently, quantitative proteomic approaches have also been applied, shedding new light on the dynamics and composition of maturing phagosomes. In this review we summarize the studies that have applied proteomic technologies to phagosomes and how they have changed our understanding of phagosome biology.
Asunto(s)
Fagocitosis/fisiología , Fagosomas/metabolismo , Proteómica , Animales , Bacterias/metabolismo , Humanos , Inmunidad Innata/fisiología , Membranas Intracelulares/metabolismo , Fagosomas/química , Vacuolas/microbiologíaRESUMEN
Macrophages use phagocytosis to control the spread of pathogens in the body, to clear apoptotic cells, and to aid in tissue remodeling. The phagosomal membrane is traditionally thought to originate from the plasmalemma and then go through a series of maturation steps involving sequential fusion with endosomal compartments, leading to the formation of a phagolysosome. A recent model suggests that the endoplasmic reticulum (ER) is involved in the maturation as well. Here we use stable isotope labeling and multiple quantitative proteomic approaches to follow the dynamic composition of the maturing phagosome in RAW 264.7 macrophage cells to a greater depth and higher temporal resolution than was previously possible. Analysis of the results suggests that the traditional model of a linear sequence of fusion events with different compartments is more complex or variable than previously thought. By concomitantly measuring the degree to which each component is enriched on phagosomes, our data argue that the amount of ER involved in phagocytosis is much less than predicted by the model of ER-mediated phagocytosis.