RESUMO
Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome-dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP-depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF-κB inhibition. Moreover, Tchp knock-out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy-mediated mechanism.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Autofagia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Centríolos/metabolismo , Células Endoteliais/metabolismo , Humanos , Camundongos , NF-kappa B , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
Cellular accumulation of reactive oxygen species (ROS) is associated with a wide range of developmental and stress responses. Although cells have evolved to use ROS as signaling molecules, their chemically reactive nature also poses a threat. Antioxidant systems are required to detoxify ROS and prevent cellular damage, but little is known about how these systems manage to function in hostile, ROS-rich environments. Here we show that during oxidative stress in plant cells, the pathogen-inducible oxidoreductase Nucleoredoxin 1 (NRX1) targets enzymes of major hydrogen peroxide (H2O2)-scavenging pathways, including catalases. Mutant nrx1 plants displayed reduced catalase activity and were hypersensitive to oxidative stress. Remarkably, catalase was maintained in a reduced state by substrate-interaction with NRX1, a process necessary for its H2O2-scavenging activity. These data suggest that unexpectedly H2O2-scavenging enzymes experience oxidative distress in ROS-rich environments and require reductive protection from NRX1 for optimal activity.
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Platinum and palladium are much sought-after metals of critical global importance in terms of abundance and availability. At the nano-scale these metals are of even higher value due to their catalytic abilities for industrial applications. Desulfovibrio alaskensis is able to capture ionic forms of both of these metals, reduce them and synthesize elemental nanoparticles. Despite this ability, very little is known about the biological pathways involved in the formation of these nanoparticles. Proteomic analysis of D. alaskensis in response to platinum and palladium has highlighted those proteins involved in both the reductive pathways and the wider stress-response system. A core set of 13 proteins was found in both treatments and consisted of proteins involved in metal transport and reduction. There were also seven proteins that were specific to either platinum or palladium. Overexpression of one of these platinum-specific genes, a NiFe hydrogenase small subunit (Dde_2137), resulted in the formation of larger nanoparticles. This study improves our understanding of the pathways involved in the metal resistance mechanism of Desulfovibrio and is informative regarding how we can tailor the bacterium for nanoparticle production, enhancing its application as a bioremediation tool and as a way to capture contaminant metals from the environment.
Assuntos
Proteínas de Bactérias/metabolismo , Desulfovibrio/metabolismo , Nanopartículas Metálicas , Paládio/metabolismo , Platina/metabolismo , Proteínas de Bactérias/genética , Biodegradação Ambiental , Desulfovibrio/genética , Hidrogenase/genética , Hidrogenase/metabolismo , Nanopartículas Metálicas/química , Modelos Biológicos , Tamanho da Partícula , ProteômicaRESUMO
Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However, C. jejuni lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery of C. jejuni proteins into host cells. Proteomic analysis of C. jejuni 11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT). C. jejuni OMVs contained 16 N-linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells. C. jejuni OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with C. jejuni OMVs. OMVs isolated from a C. jejuni 11168H cdtA mutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Campylobacter jejuni/patogenicidade , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Intestinos/microbiologia , Vesículas Transportadoras/fisiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Células CACO-2 , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/imunologia , Humanos , Interleucina-8/metabolismo , Intestinos/citologia , Intestinos/imunologia , Microscopia Eletrônica de Transmissão , Proteômica , Vesículas Transportadoras/imunologia , Vesículas Transportadoras/ultraestruturaRESUMO
Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Receptores ErbB/metabolismo , Mutação/genética , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular Tumoral , Humanos , Fosforilação , Prognóstico , Análise de Sobrevida , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
The performances of five different ESI sources coupled to a polystyrene-divinylbenzene monolithic column were compared in a series of LC-ESI-MS/MS analyses of Escherichia coli outer membrane proteins. The sources selected for comparison included two different modifications of the standard electrospray source, a commercial low-flow sprayer, a stainless steel nanospray needle and a coated glass Picotip. Respective performances were judged on sensitivity and the number and reproducibility of significant protein identifications obtained through the analysis of multiple identical samples. Data quality varied between that of a ground silica capillary, with 160 total protein identifications, the lowest number of high quality peptide hits obtained (3012), and generally peaks of lower intensity; and a stainless steel nanospray needle, which resulted in increased precursor ion abundance, the highest-quality peptide fragmentation spectra (5414) and greatest number of total protein identifications (259) exhibiting the highest MASCOT scores (average increase in score of 27.5% per identified protein). The data presented show that, despite increased variability in comparative ion intensity, the stainless steel nanospray needle provides the highest overall sensitivity. However, the resulting data were less reproducible in terms of proteins identified in complex mixtures -- arguably due to an increased number of high intensity precursor ion candidates.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização por Electrospray/normas , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/química , Peptídeos/análiseRESUMO
The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in susceptible ungulates but infects its natural host, wildebeest, without obvious clinical signs. In tissue culture, AlHV-1 is initially predominantly cell associated and virulent but on extended culture becomes cell-free and attenuated. We wanted to determine what changes in protein composition had taken place during the transition from virulent to attenuated virus in culture. Purified virus preparations were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. Peptides were identified in serial gel slices by using MASCOT software to interrogate virus-specific and nonredundant sequence databases. Twenty-three AlHV-1-encoded proteins and six cellular proteins were identified in the attenuated and virulent viruses. Two polypeptides were detected in only the virulent virus preparations, while one other protein was found in only the attenuated virus. Two of these virus-specific proteins were identified by a single peptide, suggesting that these may be low-abundance virion proteins rather than markers of attenuation or pathogenesis. The results suggest that attenuation of AlHV-1 is not the result of gross changes in the composition of the virus particle but probably due to altered viral gene expression in the infected cell.
Assuntos
Gammaherpesvirinae/metabolismo , Gammaherpesvirinae/patogenicidade , Proteômica , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Bovinos , Linhagem Celular , Gammaherpesvirinae/química , Glicoproteínas/química , Glicoproteínas/metabolismo , Espectrometria de Massas , Coelhos , Receptores Virais/metabolismo , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Vírion/química , Vírion/metabolismoRESUMO
The malaria mosquito, Anopheles stephensi, and other mosquitoes modulate their biology to match the time-of-day. In the present work, we used a non-hypothesis driven approach (untargeted proteomics) to identify proteins in mosquito tissue, and then quantified the relative abundance of the identified proteins from An. stephensi bodies. Using these quantified protein levels, we then analyzed the data for proteins that were only detectable at certain times-of-the day, highlighting the need to consider time-of-day in experimental design. Further, we extended our time-of-day analysis to look for proteins which cycle in a rhythmic 24-hour ("circadian") manner, identifying 31 rhythmic proteins. Finally, to maximize the utility of our data, we performed a proteogenomic analysis to improve the genome annotation of An. stephensi. We compare peptides that were detected using mass spectrometry but are 'missing' from the An. stephensi predicted proteome, to reference proteomes from 38 other primarily human disease vector species. We found 239 such peptide matches and reveal that genome annotation can be improved using proteogenomic analysis from taxonomically diverse reference proteomes. Examination of 'missing' peptides revealed reading frame errors, errors in gene-calling, overlapping gene models, and suspected gaps in the genome assembly.
Assuntos
Anopheles/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteogenômica/métodos , Animais , Anopheles/genética , Humanos , Índia , Proteínas de Insetos/química , Malária/transmissão , Espectrometria de Massas , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Peptídeos/análise , Proteômica/métodos , Análise de Sequência de DNARESUMO
Data are presented on the identification and partial characterisation of proteins comprising the chlamydial outer membrane complex (COMC) fraction of Chlamydia abortus (C. abortus)-the aetiological agent of ovine enzootic abortion. Inoculation with the COMC fraction is known to be highly effective in protecting sheep against experimental challenge and its constituent proteins are therefore of interest as potential vaccine candidates. Sodium N-lauroylsarcosine (sarkosyl) insoluble COMC proteins resolved by SDS-PAGE were interrogated by mass spectrometry using combined rapid monolithic column liquid chromatography and fast MS/MS scanning. Downstream database mining of processed tandem MS data revealed the presence of 67 proteins in total, including putative membrane associated proteins (n = 36), such as porins, polymorphic membrane proteins (Pmps), chaperonins and hypothetical membrane proteins, in addition to others (n = 22) that appear more likely to have originated from other subcellular compartments. Electrophoretic mobility data combined with detailed amino acid sequence information derived from secondary fragmentation spectra for 8 Pmps enabled peptides originating from protein cleavage fragments to be mapped to corresponding regions of parent precursor molecules yielding preliminary evidence in support of endogenous post-translational processing of outer membrane proteins in C. abortus. The data presented here will facilitate a deeper understanding of the pathogenesis of C. abortus infection and represent an important step towards the elucidation of the mechanisms of immunoprotection against C. abortus infection and the identification of potential target vaccine candidate antigens.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia/metabolismo , Cromatografia Líquida/métodos , Proteoma/análise , Espectrometria de Massas em Tandem/métodos , Animais , Chlamydia/isolamento & purificação , Infecções por Chlamydia/microbiologia , Feminino , Gravidez , OvinosRESUMO
Lymph node cannulation allows the collection of lymph draining from a defined anatomical region. Proteomic analysis of that lymph offers a potentially valuable insight into the immunoinflammatory response of that particular region. In this study, ovine gastric lymph has been used to monitor the proteomic changes occurring in the tissue fluid of the abomasum, in response to infection with the parasitic nematode, Teladorsagia circumcincta. Lymph, collected temporally over an experimental infection period, was analysed by means of 2-DE and subsequent gel analysis using densitometry software. In addition, the composition of the lymphatic proteome was further explored by means of MALDI-TOF and MS/MS analyses. The concentration of gelsolin, alpha-1 beta glycoprotein and haemopexin were altered significantly (p<0.05) with infection.
Assuntos
Helmintos/metabolismo , Linfonodos/parasitologia , Linfa/parasitologia , Nematoides/metabolismo , Infecções por Nematoides/metabolismo , Infecções por Nematoides/veterinária , Animais , Cromatografia Líquida de Alta Pressão/métodos , Densitometria/métodos , Eletroforese em Gel Bidimensional , Sistema Imunitário , Imunoglobulina G/química , Espectrometria de Massas/métodos , Proteômica/métodos , Ovinos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Whole cell MALDI is regularly used for the identification of bacteria to species level in clinical Microbiology laboratories. However, there remains a need to rapidly characterize and differentiate isolates below the species level to support outbreak management. We describe the implementation of a modified preparative approach for MALDI-MS combined with a custom analytical computational pipeline as a rapid procedure for subtyping Shigatoxigenic E. coli (STEC) and accurately identifying strain-specifying biomarkers. The technique was able to differentiate E. coli O157:H7 from other STEC. Within O157 serotype O157:H7 isolates were readily distinguishable from Sorbitol Fermenting O157 isolates. Overall, nine homogeneous groups of isolates were distinguished, each exhibiting distinct profiles of defining mass spectra features. This offers a robust analytical tool useable in reference/diagnostic public health scenarios.
Assuntos
Técnicas de Tipagem Bacteriana/estatística & dados numéricos , Escherichia coli O157/isolamento & purificação , Escherichia coli Shiga Toxigênica/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Técnicas de Tipagem Bacteriana/métodos , Análise de Componente Principal , Sorogrupo , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/estatística & dados numéricos , Fatores de TempoRESUMO
In plants, the circadian clock regulates the expression of one-third of all transcripts and is crucial to virtually every aspect of metabolism and growth. We now establish sumoylation, a posttranslational protein modification, as a novel regulator of the key clock protein CCA1 in the model plant Arabidopsis. Dynamic sumoylation of CCA1 is observed in planta and confirmed in a heterologous expression system. To characterize how sumoylation might affect the activity of CCA1, we investigated the properties of CCA1 in a wild-type plant background in comparison with ots1 ots2, a mutant background showing increased overall levels of sumoylation. Neither the localization nor the stability of CCA1 was significantly affected. However, binding of CCA1 to a target promoter was significantly reduced in chromatin-immunoprecipitation experiments. In vitro experiments using recombinant protein revealed that reduced affinity to the cognate promoter element is a direct consequence of sumoylation of CCA1 that does not require any other factors. Combined, these results suggest sumoylation as a mechanism that tunes the DNA binding activity of the central plant clock transcription factor CCA1.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , DNA/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC-ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith-Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.
RESUMO
Lawsonia intracellularis is the aetiological agent of the commercially significant porcine disease, proliferative enteropathy. Current understanding of host-pathogen interaction is limited due to the fastidious microaerophilic obligate intracellular nature of the bacterium. In the present study, expression of bacterial proteins during infection was investigated using a mass spectrometry approach. LC-ESI-MS/MS analysis of two isolates of L. intracellularis from heavily-infected epithelial cell cultures and database mining using fully annotated L. intracellularis genome sequences identified 19 proteins. According to the Clusters of Orthologous Groups (COG) functional classification, proteins were identified with roles in cell metabolism, protein synthesis and oxidative stress protection; seven proteins with putative or unknown function were also identified. Detailed bioinformatic analyses of five uncharacterised proteins, which were expressed by both isolates, identified domains and motifs common to other outer membrane-associated proteins with important roles in pathogenesis including adherence and invasion. Analysis of recombinant proteins on Western blots using immune sera from L. intracellularis-infected pigs identified two proteins, LI0841 and LI0902 as antigenic. The detection of five outer membrane proteins expressed during infection, including two antigenic proteins, demonstrates the potential of this approach to interrogate L. intracellularis host-pathogen interactions and identify novel targets which may be exploited in disease control.
Assuntos
Infecções por Desulfovibrionaceae/veterinária , Genoma Bacteriano/genética , Enteropatias/veterinária , Lawsonia (Bactéria)/metabolismo , Proteômica , Doenças dos Suínos/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biologia Computacional , Infecções por Desulfovibrionaceae/imunologia , Infecções por Desulfovibrionaceae/metabolismo , Infecções por Desulfovibrionaceae/virologia , Interações Hospedeiro-Patógeno , Enteropatias/microbiologia , Lawsonia (Bactéria)/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Suínos , Espectrometria de Massas em TandemRESUMO
Malignant catarrhal fever (MCF) is a fatal disease of cattle and other ungulates caused by certain gamma-herpesviruses including alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2). An attenuated virus vaccine based on AlHV-1 has been shown to induce virus-neutralising antibodies in plasma and nasal secretions of protected cattle but the targets of virus-specific antibodies are unknown. Proteomic analysis and western blotting of virus extracts allowed the identification of eight candidate AlHV-1 virion antigens. Recombinant expression of selected candidates and their OvHV-2 orthologues confirmed that two polypeptides, the products of the ORF17.5 and ORF65 genes, were antigens recognised by antibodies from natural MCF cases or from AlHV-1 vaccinated cattle. These proteins have potential as diagnostic and/or vaccine antigens.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/sangue , Proteínas do Capsídeo/imunologia , Infecções por Herpesviridae/veterinária , Herpesviridae/imunologia , Febre Catarral Maligna/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Western Blotting , Proteínas do Capsídeo/genética , Bovinos , Herpesviridae/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Febre Catarral Maligna/prevenção & controle , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Atenuadas/imunologia , Vacinas Virais/administração & dosagem , Vírion/imunologiaRESUMO
Investigation of antigenic determinants of the microaerophilic obligate intracellular bacterium Lawsonia intracellularis using a mass spectrometry approach identified a novel bacterial protein present in an extract of cell culture medium aspirated from heavily infected in vitro cell cultures. Western immunoblotting analysis of SDS-PAGE-resolved proteins using immune sera pooled from L. intracellularis-infected pigs revealed the presence of a strongly immunoreactive band of â¼ 72 kDa. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of this component and database mining using a fully annotated L. intracellularis genome sequence and the comprehensive GenBank prokaryotic genomic database highlighted the presence of a protein that shares little sequence similarity with other prokaryotic proteins and appears to be highly species specific. Detailed bioinformatic analyses identified the protein as member of the autotransporter protein family of surface-exposed proteins, and the designation LatA (Lawsonia autotransporter protein A) is suggested. Recognition of recombinant LatA on Western blots by a panel of sera from infected and control pigs corresponded 100% with a commercial serodiagnostic that relies on in vitro culture of this fastidious organism. LatA therefore represents a potential candidate for the development of a rapid and species-specific serodiagnostic reagent.