RESUMEN
The oral cavity is a habitat for different microorganisms, of which bacteria are best described. Studying different bacterial taxa and their proteins is crucial to understanding their interactions with the host and other microbes. Also, for bacteria with virulence potential, identifying novel antigenic proteins is essential to finding candidates for the development of vaccines.Here, a workflow for gel-free and label-free protein analysis of oral bacterial species grown in vitro as a biofilm and a planktonic culture is described. Details on cultivation, protein extraction and digestion, peptide cleanup, LC-MS/MS run parameters, and subsequent bioinformatics analysis are included. Challenging steps in the workflow, such as growing different types of bacteria and selecting a suitable protein database, are also discussed. This protocol provides a valuable guide for metaproteomic experiments using multi-species models of oral bacteria.
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Bacterias , Proteínas Bacterianas , Boca , Proteómica , Espectrometría de Masas en Tándem , Proteómica/métodos , Boca/microbiología , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodos , Proteínas Bacterianas/metabolismo , Humanos , Bacterias/metabolismo , Microbiota , Biopelículas/crecimiento & desarrollo , Biología Computacional/métodos , Proteoma , Flujo de TrabajoRESUMEN
Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.
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Oxigenasas de Función Mixta , Neumonía , Humanos , Ratones , Animales , Oxigenasas de Función Mixta/metabolismo , Pseudomonas aeruginosa/metabolismo , Polisacáridos/metabolismo , InmunizaciónRESUMEN
Merkel cell polyomavirus (MCPyV) is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. MCPyV large T-antigen (LTag) and small T-antigen (sTag) are the main oncoproteins involved in MCPyV-induced MCC. A hallmark of MCPyV-positive MCC cells is the expression of a C-terminal truncated LTag. Protein kinase A (PKA) plays a fundamental role in a variety of biological processes, including transcription by phosphorylating and thereby regulating the activity of transcription factors. As MCPyV LTag has been shown to be phosphorylated and acts as a transcription factor for the viral early and late promoter, we investigated whether LTag can be phosphorylayted by PKA, and whether this affects the transcript activity of LTag. Using a phosphorylation prediction algorithm, serine 191, 203, and 265 were identified as putative phosphorylation sites for PKA. Mass spectrometry of in vitro PKA-phosphorylated peptides confirmed phosphorylation of S203 and S265, but not S191. Full-length LTag inhibited early and late promoter activity of MCPyV, whereas the truncated MKL2 LTag variant stimulated both promoters. Single non-phosphorylable, as well as phosphomimicking mutations did not alter the inhibitory effect of full-length LTag. However, the non-phosphorylable mutations abrogated transactivation of the MCPyV promoters by MKL2 LTag, whereas phosphomimicking substitutions restored the ability of MKL2 LTag to activate the promoters. Triple LTag and MKL2 LTag mutants had the same effect as the single mutants. Activation of the PKA signaling pathway did not enhance MCPyV promoter activity, nor did it affect LTag expression levels in MCPyV-positive Merkel cell carcinoma (MCC) cells. Our results show that phosphorylation of truncated LTag stimulates viral promoter activity, which may contribute to higher levels of the viral oncoproteins LTag and sTag. Interfering with PKA-induced LTag phosphorylation/activity may be a therapeutic strategy to treat MCPyV-positive MCC patients.
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Antígenos Transformadores de Poliomavirus , Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Infecciones Tumorales por Virus , Humanos , Carcinoma de Células de Merkel/metabolismo , Carcinoma de Células de Merkel/virología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Poliomavirus de Células de Merkel/metabolismo , Fosforilación , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/virología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virología , Antígenos Transformadores de Poliomavirus/metabolismo , Transcripción GenéticaRESUMEN
INTRODUCTION: Liver sinusoidal endothelial cells (LSECs) are specialized fenestrated scavenger endothelial cells involved in the elimination of modified plasma proteins and tissue turnover waste macromolecules from blood. LSECs also participate in liver immune responses. A challenge when studying LSEC biology is the rapid loss of the in vivo phenotype in culture. In this study, we have examined biological processes and pathways affected during early-stage primary culture of rat LSECs and checked for cell responses to the pro-inflammatory cytokine interleukin (IL)-1ß and the anti-inflammatory drug dexamethasone. METHODS: LSECs from male Sprague Dawley rats were cultured on type I collagen in 5% oxygen atmosphere in DMEM with serum-free supplements for 2 and 24 h. Quantitative proteomics using tandem mass tag technology was used to examine proteins in cells and supernatants. Validation was done with qPCR, ELISA, multiplex immunoassay, and caspase 3/7 assay. Cell ultrastructure was examined by scanning electron microscopy, and scavenger function by quantitative endocytosis assays. RESULTS: LSECs cultured for 24 h showed a characteristic pro-inflammatory phenotype both in the presence and absence of IL-1ß, with upregulation of cellular responses to cytokines and interferon-γ, cell-cell adhesion, and glycolysis, increased expression of fatty acid binding proteins (FABP4, FABP5), and downregulation of several membrane receptors (STAB1, STAB2, LYVE1, CLEC4G) and proteins in pyruvate metabolism, citric acid cycle, fatty acid elongation, amino acid metabolism, and oxidation-reduction processes. Dexamethasone inhibited apoptosis and improved LSEC viability in culture, repressed inflammatory and immune regulatory pathways and secretion of IL-1ß and IL-6, and further upregulated FABP4 and FABP5 compared to time-matched controls. The LSEC porosity and endocytic activity were reduced at 24 h both with and without dexamethasone but the dexamethasone-treated cells showed a less stressed phenotype. CONCLUSION: Rat LSECs become activated towards a pro-inflammatory phenotype during early culture. Dexamethasone represses LSEC activation, inhibits apoptosis, and improves cell viability.
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Células Endoteliales , Proteoma , Animales , Dexametasona/metabolismo , Dexametasona/farmacología , Células Endoteliales/metabolismo , Hígado/metabolismo , Masculino , Proteoma/metabolismo , Ratas , Ratas Sprague-Dawley , SecretomaRESUMEN
Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.
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Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Células HEK293 , Células HeLa , Humanos , Mamíferos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Unión Proteica , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The Golgi complex is essential for the processing, sorting, and trafficking of newly synthesized proteins and lipids. Golgi turnover is regulated to meet different cellular physiological demands. The role of autophagy in the turnover of Golgi, however, has not been clarified. Here we show that CALCOCO1 binds the Golgi-resident palmitoyltransferase ZDHHC17 to facilitate Golgi degradation by autophagy during starvation. Depletion of CALCOCO1 in cells causes expansion of the Golgi and accumulation of its structural and membrane proteins. ZDHHC17 itself is degraded by autophagy together with other Golgi membrane proteins such as TMEM165. Taken together, our data suggest a model in which CALCOCO1 mediates selective Golgiphagy to control Golgi size and morphology in eukaryotic cells via its interaction with ZDHHC17.
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Aciltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Proteínas de Unión al Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción/metabolismo , Aciltransferasas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Unión al Calcio/genética , Aparato de Golgi/genética , Células HeLa , Humanos , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas , Factores de Transcripción/genéticaRESUMEN
The endoplasmic reticulum (ER) plays important roles in protein synthesis and folding, and calcium storage. The volume of the ER and expression of its resident proteins are increased in response to nutrient stress. ER-phagy, a selective form of autophagy, is involved in the degradation of the excess components of the ER to restore homeostasis. Six ER-resident proteins have been identified as ER-phagy receptors so far. In this study, we have identified CALCOCO1 as a novel ER-phagy receptor for the degradation of the tubular ER in response to proteotoxic and nutrient stress. CALCOCO1 is a homomeric protein that binds directly to ATG8 proteins via LIR- and UDS-interacting region (UIR) motifs acting co-dependently. CALCOCO1-mediated ER-phagy requires interaction with VAMP-associated proteins VAPA and VAPB on the ER membranes via a conserved FFAT-like motif. Depletion of CALCOCO1 causes expansion of the ER and inefficient basal autophagy flux. Unlike the other ER-phagy receptors, CALCOCO1 is peripherally associated with the ER. Therefore, we define CALCOCO1 as a soluble ER-phagy receptor.
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Autofagia , Proteínas de Unión al Calcio/metabolismo , Membranas Intracelulares/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Ratones , Factores de Transcripción/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase Cζ (PKCζ) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.
Asunto(s)
Autofagia/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Quinasas Relacionadas con NIMA/metabolismo , Dominios y Motivos de Interacción de Proteínas/genética , Proteína Sequestosoma-1/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Cromatografía Líquida de Alta Presión , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Mutación , Quinasas Relacionadas con NIMA/genética , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño , Proteína Sequestosoma-1/química , Proteína Sequestosoma-1/genética , Serina-Treonina Quinasa 3 , Espectrometría de Masas en Tándem , Treonina/metabolismoRESUMEN
The mechanism of action of recombinant IgG2/4 antibodies involves blocking of their target without the induction of effector functions. Examples are eculizumab (Soliris®), which is used clinically to block complement factor C5, as well as anti-human CD14 (r18D11) and anti-porcine CD14 (rMIL2) produced in our laboratory. So far, no proper IgG2/4 control antibody has been available for controlled validation of IgG2/4 antibody functions. Here, we describe the design of a recombinant control antibody (NHDL), which was generated by combining the variable light (VL) and heavy (VH) chains from two unrelated specificities. NHDL was readily expressed and purified as a stable IgG2/4 antibody, and showed no detectable specificity toward any putative antigen present in human or porcine blood. The approach of artificial VL/VH combination may be adopted for the design of other recombinant control antibodies.
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Anticuerpos Monoclonales/genética , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Proteínas Recombinantes de Fusión/genética , Animales , Anticuerpos Monoclonales/metabolismo , Terapia Biológica , Proteínas Sanguíneas/metabolismo , Epítopos/metabolismo , Humanos , Receptores de Lipopolisacáridos/inmunología , Receptores de Lipopolisacáridos/metabolismo , Ratones , Placebos , Ingeniería de Proteínas , PorcinosRESUMEN
Syntaxin 17 (Stx17) has been implicated in autophagosome-lysosome fusion. Here, we report that Stx17 functions in assembly of protein complexes during autophagy initiation. Stx17 is phosphorylated by TBK1 whereby phospho-Stx17 controls the formation of the ATG13+FIP200+ mammalian pre-autophagosomal structure (mPAS) in response to induction of autophagy. TBK1 phosphorylates Stx17 at S202. During autophagy induction, Stx17pS202 transfers from the Golgi, where its steady-state pools localize, to the ATG13+FIP200+ mPAS. Stx17pS202 was in complexes with ATG13 and FIP200, whereas its non-phosphorylatable mutant Stx17S202A was not. Stx17 or TBK1 knockouts blocked ATG13 and FIP200 puncta formation. Stx17 or TBK1 knockouts reduced the formation of ATG13 protein complexes with FIP200 and ULK1. Endogenous Stx17pS202 colocalized with LC3B following induction of autophagy. Stx17 knockout diminished LC3 response and reduced sequestration of the prototypical bulk autophagy cargo lactate dehydrogenase. We conclude that Stx17 is a TBK1 substrate and that together they orchestrate assembly of mPAS.
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Autofagia/genética , Complejos Multiproteicos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Qa-SNARE/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Técnicas de Inactivación de Genes , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisosomas/metabolismo , Fusión de Membrana/genética , Complejos Multiproteicos/metabolismo , Mutación/genética , Fosforilación , Proteínas Tirosina Quinasas/genética , Transducción de Señal/genéticaRESUMEN
Bacterial membrane vesicles (MVs) mediate bacterial virulence by enabling secretion and long distance delivery of bacterial effector molecules. Staphylococcus haemolyticus has now been demonstrated to produce membrane vesicles (MVs). The protein content of S. haemolyticus MVs was identified by Mass spectrometry and compared to proteins identified in the total secretome. This information is presented in this data article. Further background and interpretation of the data can be found in the article: Comparative exoproteome profiling of an invasive and a commensal S. haemolyticus isolate (Cavanagh et al., in press). Data are available via Proteome Xchange with identifier PXD010389.
RESUMEN
Fish immunology research is at a pivotal point with the increasing availability of functional immunoassays and major advances in omics approaches. However, studies on fish B cells and their distinct subsets remain a challenge due to the limited availability of differentially expressed surface markers. To address this constraint, cell surface proteome of Atlantic salmon IgM+ B cells were analyzed by mass spectrometry and compared to surface proteins detected from two adherent salmon head kidney cell lines, ASK and SSP-9. Out of 21 cluster of differentiation (CD) molecules identified on salmon IgM+ B cells, CD22 and CD79A were shortlisted as potential markers based on the reported B cell-specific surface expression of their mammalian homologs. Subsequent RT-qPCR analyses of flow cytometry-sorted subpopulations from head kidney leukocytes confirmed that both cd22 and cd79a genes were highly expressed in IgM+ lymphoid cells but were observed in barely detectable levels in IgM- non-lymphoid suspension and adherent cells. Similarly, significantly high cd22 and cd79a mRNA levels were observed in IgM+ or IgT+ lymphoid cells from the spleen and peritoneal cavity, but not in their corresponding IgM- IgT- non-lymphoid fractions. This suggests that the B cell restrictive expression of CD22 and CD79A extend down to the transcription level, which was consistent across different lymphoid compartments and immunoglobulin isotypes, thus strongly supporting the potential of CD22 and CD79A as pan-B cell markers for salmon. In addition, this study provides novel information on the salmon B cell surface protein repertoire, as well as insights on B cell evolution. Further investigation of the identified salmon CD molecules, including development of immunological tools for detection, will help advance our understanding of the dynamics of salmon B cell responses such as during infection, vaccination, or immunostimulation.
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Linfocitos B/inmunología , Linfocitos B/metabolismo , Inmunoglobulina M/inmunología , Proteínas de la Membrana/metabolismo , Proteoma , Salmo salar/inmunología , Salmo salar/metabolismo , Animales , Células Cultivadas , Proteínas de Peces/metabolismo , Perfilación de la Expresión Génica , Espectrometría de Masas , Proteínas de la Membrana/genética , Proteómica/métodos , Salmo salar/genéticaRESUMEN
Resin-based biomaterials are widely used in medical and dental treatment, and both clinicians and patients are exposed to the materials. The knowledge of toxicity is mainly based on in vitro studies at exposure concentrations that induce cell death. However, severe cell damage and cell death signaling may overshadow essential cellular events caused by a possible toxicant. For dental resins, the knowledge of interaction with living cells at more clinical relevant exposure doses is sparse. 2-Hydroxyethylmethacrylate (HEMA) is a commonly used monomer in dental resins. Measuring cellular adaptation to HEMA at concentrations that did not reduce cell viability was the main focus of this study. Stable isotope labeling with amino acids in cell culture was used to measure proteome changes in cultured THP-1 cells exposed to HEMA. Western blotting verified the results. Cells exposed to HEMA increased their level of several cytoprotective proteins. The observed adaptation is compatible with increased oxidative burden caused by GSH depletion and the electrophilic characteristic of HEMA. The present approach to analyzing the toxic potential of HEMA yielded information on interactions with living cells is not previously reported. This detailed information is of great value to make better predictions of possible side effects in the clinic. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 851-859, 2019.
Asunto(s)
Metacrilatos/farmacología , Proteoma/metabolismo , Resinas Sintéticas/farmacología , Humanos , Células THP-1RESUMEN
Staphylococcus haemolyticus is a skin commensal emerging as an opportunistic pathogen. Nosocomial isolates of S. haemolyticus are the most antibiotic resistant members of the coagulase negative staphylococci (CoNS), but information about other S. haemolyticus virulence factors is scarce. Bacterial membrane vesicles (MVs) are one mediator of virulence by enabling secretion and long distance delivery of bacterial effector molecules while protecting the cargo from proteolytic degradation from the environment. We wanted to determine if the MV protein cargo of S. haemolyticus is strain specific and enriched in certain MV associated proteins compared to the totalsecretome. The present study shows that both clinical and commensal S. haemolyticus isolates produce membrane vesicles. The MV cargo of both strains was enriched in proteins involved in adhesion and acquisition of iron. The MV cargo of the clinical strain was further enriched in antimicrobial resistance proteins. Data are available via ProteomeXchange with identifier PXD010389. BIOLOGICAL SIGNIFICANCE: Clinical isolates of Staphylococcus haemolyticus are usually multidrug resistant, their main virulence factor is formation of biofilms, both factors leading to infections that are difficult to treat. We show that both clinical and commensal S. haemolyticus isolates produce membrane vesicles. Identification of staphylococcal membrane vesicles can potentially be used in novel approaches to combat staphylococcal infections, such as development of vaccines.
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Proteínas Bacterianas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Bases de Datos de Proteínas , Proteínas de la Membrana/metabolismo , Proteómica , Staphylococcus haemolyticus/metabolismo , Humanos , Staphylococcus haemolyticus/aislamiento & purificaciónRESUMEN
It is not clear to what extent starvation-induced autophagy affects the proteome on a global scale and whether it is selective. In this study, we report based on quantitative proteomics that cells during the first 4 h of acute starvation elicit lysosomal degradation of up to 2-3% of the proteome. The most significant changes are caused by an immediate autophagic response elicited by shortage of amino acids but executed independently of mechanistic target of rapamycin and macroautophagy. Intriguingly, the autophagy receptors p62/SQSTM1, NBR1, TAX1BP1, NDP52, and NCOA4 are among the most efficiently degraded substrates. Already 1 h after induction of starvation, they are rapidly degraded by a process that selectively delivers autophagy receptors to vesicles inside late endosomes/multivesicular bodies depending on the endosomal sorting complex required for transport III (ESCRT-III). Our data support a model in which amino acid deprivation elicits endocytosis of specific membrane receptors, induction of macroautophagy, and rapid degradation of autophagy receptors by endosomal microautophagy.
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Autofagia , Endosomas/metabolismo , Modelos Biológicos , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coactivadores de Receptor Nuclear/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas de Unión al ARN/genética , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismoRESUMEN
Triethylene glycol dimethacrylate (TEGDMA) is commonly used in polymer resin-based dental materials. This study investigated the molecular mechanisms of TEGDMA toxicity by identifying its time- and dose-dependent effects on the proteome of human THP-1 monocytes. The effects of different concentrations (0.07-5 mM) and exposure times (0-72 h) of TEGDMA on cell viability, proliferation, and morphology were determined using a real-time viability assay, automated cell counting, and electron microscopy, and laid the fundament for choice of exposure scenarios in the proteomic experiments. Solvents were not used, as TEGDMA is soluble in cell culture medium (determined by photon correlation spectroscopy). Cells were metabolically labeled [using the stable isotope labeled amino acids in cell culture (SILAC) strategy], and exposed to 0, 0.3 or 2.5 mM TEGDMA for 6 or 16 h before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Regulated proteins were analyzed in the STRING database. Cells exposed to 0.3 mM TEGDMA showed increased viability and time-dependent upregulation of proteins associated with stress/oxidative stress, autophagy, and cytoprotective functions. Cells exposed to 2.5 mM TEGDMA showed diminished viability and a protein expression profile associated with oxidative stress, DNA damage, mitochondrial dysfunction, and cell cycle inhibition. Altered expression of immune genes was observed in both groups. The study provides novel knowledge about TEGDMA toxicity at the proteomic level. Of note, even low doses of TEGDMA induced a substantial cellular response.
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Monocitos/efectos de los fármacos , Polietilenglicoles/toxicidad , Ácidos Polimetacrílicos/toxicidad , Proteoma , Células THP-1/efectos de los fármacos , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida , Daño del ADN , Materiales Dentales , Relación Dosis-Respuesta a Droga , Humanos , Ensayo de Materiales , Mitocondrias/efectos de los fármacos , Estrés Oxidativo , Especies Reactivas de Oxígeno , Solventes , Espectrometría de Masas en TándemRESUMEN
Selective autophagy performs an array of tasks to maintain intracellular homeostasis, sterility, and organellar and cellular functionality. The fidelity of these processes depends on precise target recognition and limited activation of the autophagy apparatus in a localized fashion. Here we describe cooperation in such processes between the TRIM family and Galectin family of proteins. TRIMs, which are E3 ubiquitin ligases, displayed propensity to associate with Galectins. One specific TRIM, TRIM16, interacted with Galectin-3 in a ULK1-dependent manner. TRIM16, through integration of Galectin- and ubiquitin-based processes, coordinated recognition of membrane damage with mobilization of the core autophagy regulators ATG16L1, ULK1, and Beclin 1 in response to damaged endomembranes. TRIM16 affected mTOR, interacted with TFEB, and influenced TFEB's nuclear translocation. The cooperation between TRIM16 and Galectin-3 in targeting and activation of selective autophagy protects cells from lysosomal damage and Mycobacterium tuberculosis invasion.
Asunto(s)
Autofagia , Proteínas de Unión al ADN/metabolismo , Galectina 3/metabolismo , Homeostasis , Membranas Intracelulares/metabolismo , Factores de Transcripción/metabolismo , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Beclina-1/metabolismo , Calcineurina/metabolismo , Citoprotección , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/metabolismo , Lisosomas/patología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Mycobacterium tuberculosis/fisiología , Fosforilación , Unión Proteica , Estabilidad Proteica , Células RAW 264.7 , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
The minor capsid protein of human BK polyomavirus (BKPyV), VP2, and its N-terminally truncated form, VP3, are both important for viral entry. The closely related simian virus 40 (SV40) reportedly produces an additional truncated form of VP2/3, denoted VP4, apparently functioning as a viroporin promoting progeny release. The VP4 open reading frame is conserved in some polyomaviruses, including BKPyV. In this study, we investigated the role of VP4 in BKPyV replication. By transfecting viral genomes into primary human renal proximal tubule epithelial cells, we demonstrated that unaltered BKPyV and mutants with start codon substitutions in VP4 (VP2M229I and VP2M229A) abolishing putative VP4 production were released at the same level to supernatants. However, during infection studies, VP2M229I and VP2M229A exhibited 90% and 65% reduced infectivity, respectively, indicating that isoleucine substitution inadvertently disrupted VP2/3 function to the detriment of viral entry, while inhibition of VP4 production during late infection was well tolerated. Unexpectedly, and similarly to BKPyV, wild-type SV40 and the corresponding VP4 start codon mutants (VP2M228I and VP2M228A) transfected into monkey kidney cell lines were also released at equal levels. Upon infection, only the VP2M228I mutant exhibited reduced infectivity, a 43% reduction, which also subsequently led to delayed host cell lysis. Mass spectrometry analysis of nuclear extracts from SV40-infected cells failed to identify VP4. Our results suggest that neither BKPyV nor SV40 require VP4 for progeny release. Moreover, our results reveal an important role in viral entry for the amino acid in VP2/VP3 unavoidably changed by VP4 start codon mutagenesis. IMPORTANCE: Almost a decade ago, SV40 was reported to produce a late nonstructural protein, VP4, which forms pores in the nuclear membrane, facilitating progeny release. By performing transfection studies with unaltered BKPyV and SV40 and their respective VP4-deficient mutants, we found that VP4 is dispensable for progeny release, contrary to the original findings. However, infection studies demonstrated a counterintuitive reduction of infectivity of certain VP4-deficient mutants. In addition to the isoleucine-substituted SV40 mutant of the original study, we included alanine-substituted VP4-deficient mutants of BKPyV (VP2M229A) and SV40 (VP2M228A). These revealed that the reduction in infectivity was not caused by a lack of VP4 but rather depended on the identity of the single amino acid substituted within VP2/3 for VP4 start codon mutagenesis. Hopefully, our results will correct the longstanding misconception of VP4's role during infection and stimulate continued work on unraveling the mechanism for release of polyomavirus progeny.
Asunto(s)
Virus BK/genética , Infecciones por Polyomavirus/virología , Poliomavirus/genética , Virus 40 de los Simios/genética , Sustitución de Aminoácidos/genética , Animales , Células COS , Proteínas de la Cápside/genética , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Replicación del ADN/genética , Células HeLa , Humanos , Células Vero , Internalización del Virus , Replicación Viral/genéticaRESUMEN
Extracellular vesicles or exosomes constitute an evolutionarily conserved mechanism of intercellular signaling. Exosomes are gaining an increasing amount of attention due to their role in pathologies, including malignancy, their importance as prognostic and diagnostic markers, and their potential as a therapeutic tool. Merkel cell carcinoma (MCC) is an aggressive form of skin cancer with a poor prognosis. Because an effective systemic treatment for this cancer type is currently not available, an exosome-based therapy was proposed. However, comprehensive secretome profiling has not been performed for MCC. To help unveil the putative contribution of exosomes in MCC, we studied the protein content of MCC-derived exosomes. Since approximately 80% of all MCC cases contain Merkel cell polyomavirus (MCPyV), the secretomes of two MCPyV-negative and two MCPyV-positive MCC cell lines were compared. We identified with high confidence 164 exosome-derived proteins common for all four cell lines that were annotated in ExoCarta and Vesiclepedia databases. These include proteins implicated in motility, metastasis and tumor progression, such as integrins and tetraspanins, intracellular signaling molecules, chaperones, proteasomal proteins, and translation factors. Additional virus-negative and virus-positive MCC cell lines should be examined to identify highly representative exosomal proteins that may provide reliable prognostic and diagnostic biomarkers, as well as targets for treatment in the future. Data are available via ProteomeXchange with identifier PXD004198.