RESUMO
Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) (ISG15) is a ubiquitin-like modifier induced during infections and involved in host defense mechanisms. Not surprisingly, many viruses encode deISGylating activities to antagonize its effect. Here we show that infection by Zika, SARS-CoV-2 and influenza viruses induce ISG15-modifying enzymes. While influenza and Zika viruses induce ISGylation, SARS-CoV-2 triggers deISGylation instead to generate free ISG15. The ratio of free versus conjugated ISG15 driven by the papain-like protease (PLpro) enzyme of SARS-CoV-2 correlates with macrophage polarization toward a pro-inflammatory phenotype and attenuated antigen presentation. In vitro characterization of purified wild-type and mutant PLpro revealed its strong deISGylating over deubiquitylating activity. Quantitative proteomic analyses of PLpro substrates and secretome from SARS-CoV-2-infected macrophages revealed several glycolytic enzymes previously implicated in the expression of inflammatory genes and pro-inflammatory cytokines, respectively. Collectively, our results indicate that altered free versus conjugated ISG15 dysregulates macrophage responses and probably contributes to the cytokine storms triggered by SARS-CoV-2.
Assuntos
COVID-19/imunologia , Citocinas/metabolismo , Inflamação/imunologia , Macrófagos/imunologia , SARS-CoV-2/fisiologia , Ubiquitinas/metabolismo , Diferenciação Celular , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Citocinas/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Evasão da Resposta Imune , Imunidade Inata , Vírus da Influenza A/fisiologia , Influenza Humana/imunologia , Células-Tronco Pluripotentes/citologia , Ubiquitinação , Ubiquitinas/genética , Zika virus/fisiologia , Infecção por Zika virus/imunologiaRESUMO
The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane ß-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic ß-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate ß-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.
Assuntos
Proteínas da Membrana Bacteriana Externa , Membrana Celular , Bactérias Gram-Negativas , Bicamadas Lipídicas , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Glicina/metabolismo , Lipopolissacarídeos/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Fosfolipídeos/metabolismo , Sítios de Ligação , Proteostase , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteoma/química , Proteoma/metabolismo , Regulon , Domínios Proteicos , Peptídeos Antimicrobianos/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/metabolismoRESUMO
The resistance of cancer cells to therapy is responsible for the death of most patients with cancer1. Epithelial-to-mesenchymal transition (EMT) has been associated with resistance to therapy in different cancer cells2,3. However, the mechanisms by which EMT mediates resistance to therapy remain poorly understood. Here, using a mouse model of skin squamous cell carcinoma undergoing spontaneous EMT during tumorigenesis, we found that EMT tumour cells are highly resistant to a wide range of anti-cancer therapies both in vivo and in vitro. Using gain and loss of function studies in vitro and in vivo, we found that RHOJ-a small GTPase that is preferentially expressed in EMT cancer cells-controls resistance to therapy. Using genome-wide transcriptomic and proteomic profiling, we found that RHOJ regulates EMT-associated resistance to chemotherapy by enhancing the response to replicative stress and activating the DNA-damage response, enabling tumour cells to rapidly repair DNA lesions induced by chemotherapy. RHOJ interacts with proteins that regulate nuclear actin, and inhibition of actin polymerization sensitizes EMT tumour cells to chemotherapy-induced cell death in a RHOJ-dependent manner. Together, our study uncovers the role and the mechanisms through which RHOJ acts as a key regulator of EMT-associated resistance to chemotherapy.
Assuntos
Carcinoma de Células Escamosas , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Neoplasias Cutâneas , Proteínas rho de Ligação ao GTP , Actinas/efeitos dos fármacos , Actinas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteômica , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Camundongos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Perfilação da Expressão Gênica , GenomaRESUMO
The skin epidermis is constantly renewed throughout life1,2. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumour initiation3. However, the ways in which oncogenic mutations affect the balance between renewal and differentiation and lead to clonal expansion, cell competition, tissue colonization and tumour development are unknown. Here, through multidisciplinary approaches that combine in vivo clonal analysis using intravital microscopy, single-cell analysis and functional analysis, we show how SmoM2-a constitutively active oncogenic mutant version of Smoothened (SMO) that induces the development of basal cell carcinoma-affects clonal competition and tumour initiation in real time. We found that expressing SmoM2 in the ear epidermis of mice induced clonal expansion together with tumour initiation and invasion. By contrast, expressing SmoM2 in the back-skin epidermis led to a clonal expansion that induced lateral cell competition without dermal invasion and tumour formation. Single-cell analysis showed that oncogene expression was associated with a cellular reprogramming of adult interfollicular cells into an embryonic hair follicle progenitor (EHFP) state in the ear but not in the back skin. Comparisons between the ear and the back skin revealed that the dermis has a very different composition in these two skin types, with increased stiffness and a denser collagen I network in the back skin. Decreasing the expression of collagen I in the back skin through treatment with collagenase, chronic UV exposure or natural ageing overcame the natural resistance of back-skin basal cells to undergoing EHFP reprogramming and tumour initiation after SmoM2 expression. Altogether, our study shows that the composition of the extracellular matrix regulates how susceptible different regions of the body are to tumour initiation and invasion.
Assuntos
Transformação Celular Neoplásica , Matriz Extracelular , Neoplasias Cutâneas , Microambiente Tumoral , Animais , Camundongos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colágeno/metabolismo , Epiderme/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Neoplasias Cutâneas/patologia , Carcinoma Basocelular/patologia , Orelha/patologia , Colagenases/metabolismo , Envelhecimento , Raios Ultravioleta , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismoRESUMO
FAT1, which encodes a protocadherin, is one of the most frequently mutated genes in human cancers1-5. However, the role and the molecular mechanisms by which FAT1 mutations control tumour initiation and progression are poorly understood. Here, using mouse models of skin squamous cell carcinoma and lung tumours, we found that deletion of Fat1 accelerates tumour initiation and malignant progression and promotes a hybrid epithelial-to-mesenchymal transition (EMT) phenotype. We also found this hybrid EMT state in FAT1-mutated human squamous cell carcinomas. Skin squamous cell carcinomas in which Fat1 was deleted presented increased tumour stemness and spontaneous metastasis. We performed transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies, which revealed that loss of function of FAT1 activates a CAMK2-CD44-SRC axis that promotes YAP1 nuclear translocation and ZEB1 expression that stimulates the mesenchymal state. This loss of function also inactivates EZH2, promoting SOX2 expression, which sustains the epithelial state. Our comprehensive analysis identified drug resistance and vulnerabilities in FAT1-deficient tumours, which have important implications for cancer therapy. Our studies reveal that, in mouse and human squamous cell carcinoma, loss of function of FAT1 promotes tumour initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state.
Assuntos
Caderinas/deficiência , Transição Epitelial-Mesenquimal/genética , Deleção de Genes , Metástase Neoplásica/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Metástase Neoplásica/tratamento farmacológico , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Proteômica , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Quinases da Família src/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8+ T cell response, but very few T cell epitopes are known in mice as a common animal infection model for listeriosis. To identify epitopes we screened for Listeria immunopeptides presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. We mapped more than 6,000 mouse self-peptides presented on MHC Class I molecules, including 12 high confident Listeria peptides from 12 different bacterial proteins. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to activate CD8+ T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. The epitope showed high biological potency in a prime boost model and can be used as a research tool to probe CD8+ T cell responses in mouse models of Listeria infection. Together, our results demonstrate the power of immunopeptidomics for bacterial antigen identification.
RESUMO
The host-virus interactome is increasingly recognized as an important research field to discover new therapeutic targets to treat influenza. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify new pro- and antiviral host factors of the influenza A virus. However, at present, a comprehensive summary of the results is lacking. We performed a systematic review of all reported CRISPR studies in this field in combination with a meta-analysis using the algorithm of meta-analysis by information content (MAIC). Two ranked gene lists were generated based on evidence in 15 proviral and 4 antiviral screens. Enriched pathways in the proviral MAIC results were compared to those of a prior array-based RNA interference (RNAi) meta-analysis. The top 50 proviral MAIC list contained genes whose role requires further elucidation, such as the endosomal ion channel TPCN1 and the kinase WEE1. Moreover, MAIC indicated that ALYREF, a component of the transcription export complex, has antiviral properties, whereas former knockdown experiments attributed a proviral role to this host factor. CRISPR-Cas-pooled screens displayed a bias toward early-replication events, whereas the prior RNAi meta-analysis covered early and late-stage events. RNAi screens led to the identification of a larger fraction of essential genes than CRISPR screens. In summary, the MAIC algorithm points toward the importance of several less well-known pathways in host-influenza virus interactions that merit further investigation. The results from this meta-analysis of CRISPR screens in influenza A virus infection may help guide future research efforts to develop host-directed anti-influenza drugs. IMPORTANCE: Viruses rely on host factors for their replication, whereas the host cell has evolved virus restriction factors. These factors represent potential targets for host-oriented antiviral therapies. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify pro- and antiviral host factors in the context of influenza virus infection. We performed a comprehensive analysis of the outcome of these screens based on the publicly available gene lists, using the recently developed algorithm meta-analysis by information content (MAIC). MAIC allows the systematic integration of ranked and unranked gene lists into a final ranked gene list. This approach highlighted poorly characterized host factors and pathways with evidence from multiple screens, such as the vesicle docking and lipid metabolism pathways, which merit further exploration.
Assuntos
Sistemas CRISPR-Cas , Interações Hospedeiro-Patógeno , Vírus da Influenza A , Influenza Humana , Humanos , Vírus da Influenza A/genética , Influenza Humana/virologia , Influenza Humana/genética , Interações Hospedeiro-Patógeno/genética , Replicação Viral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Interferência de RNARESUMO
Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Arginina/metabolismo , Grânulos Citoplasmáticos/metabolismo , Dipeptídeos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Arginina/química , Proteína C9orf72 , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/patologia , DNA Helicases , Dipeptídeos/química , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Gotículas Lipídicas/metabolismo , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Domínios Proteicos , Proteínas/química , RNA/metabolismo , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Fatores de Tempo , TransfecçãoRESUMO
SignificanceChemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.
Assuntos
Brassinosteroides/metabolismo , Ácidos Indolacéticos/metabolismo , Proteoma , Transdução de Sinais , Aminopiridinas/metabolismo , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteômica/métodos , Succinatos/metabolismoRESUMO
Data-independent acquisition (DIA) has become a well-established method for MS-based proteomics. However, the list of options to analyze this type of data is quite extensive, and the use of spectral libraries has become an important factor in DIA data analysis. More specifically the use of in silico predicted libraries is gaining more interest. By working with a differential spike-in of human standard proteins (UPS2) in a constant yeast tryptic digest background, we evaluated the sensitivity, precision, and accuracy of the use of in silico predicted libraries in data DIA data analysis workflows compared to more established workflows. Three commonly used DIA software tools, DIA-NN, EncyclopeDIA, and Spectronaut, were each tested in spectral library mode and spectral library-free mode. In spectral library mode, we used independent spectral library prediction tools PROSIT and MS2PIP together with DeepLC, next to classical data-dependent acquisition (DDA)-based spectral libraries. In total, we benchmarked 12 computational workflows for DIA. Our comparison showed that DIA-NN reached the highest sensitivity while maintaining a good compromise on the reproducibility and accuracy levels in either library-free mode or using in silico predicted libraries pointing to a general benefit in using in silico predicted libraries.
Assuntos
Simulação por Computador , Proteômica , Software , Fluxo de Trabalho , Proteômica/métodos , Proteômica/estatística & dados numéricos , Humanos , Reprodutibilidade dos Testes , Análise de Dados , Biblioteca de PeptídeosRESUMO
With current trends in proteomics, especially regarding clinical and low input (to single cell) samples, it is increasingly important to both maximize the throughput of the analysis and maintain as much sensitivity as possible. The new generation of mass spectrometers (MS) are taking a huge leap in sensitivity, allowing analysis of samples with shorter liquid chromatography (LC) methods while digging as deep in the proteome. However, the throughput can be doubled by implementing a dual column nano-LC-MS configuration. For this purpose, we used a dual-column setup with a two-outlet electrospray source and compared it to a classic dual-column setup with a single-outlet source.
Assuntos
Nanotecnologia , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Humanos , Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala/métodosRESUMO
BACKGROUND: European beech (Fagus sylvatica L.) trees produce seeds irregularly; therefore, it is necessary to store beech seeds for forestation. Despite the acquisition of desiccation tolerance during development, beech seeds are classified as intermediate because they lose viability during long-term storage faster than typical orthodox seeds. In this study, beech seeds stored for short (3 years) or long (20 years) periods under optimal conditions and displaying 92 and 30% germination capacity, respectively, were compared. RESULTS: Aged seeds displayed increased membrane damage, manifested as electrolyte leakage and lipid peroxidation levels. Analyses have been based on embryonic axes, which contained higher levels of reactive oxygen species (ROS) and higher levels of protein-bound methionine sulfoxide (MetO) in aged seeds. Using label-free quantitative proteomics, 3,949 proteins were identified, of which 2,442 were reliably quantified pointing to 24 more abundant proteins and 35 less abundant proteins in beech seeds under long-term storage conditions. Functional analyses based on gene ontology annotations revealed that nucleic acid binding activity (molecular function), ribosome organization or biogenesis and transmembrane transport (cellular processes), translational proteins (protein class) and membranous anatomical entities (cellular compartment) were affected in aged seeds. To verify whether MetO, the oxidative posttranslational modification of proteins that can be reversed via the action of methionine sulfoxide reductase (Msr) enzymes, is involved in the aging of beech seeds, we identified and quantified 226 MetO-containing proteins, among which 9 and 19 exhibited significantly up- and downregulated MetO levels, respectively, in beech seeds under long-term storage conditions. Several Msr isoforms were identified and recognized as MsrA1-like, MsrA4, MsrB5 and MsrB5-like in beech seeds. Only MsrA1-like displayed decreased abundance in aged seeds. CONCLUSIONS: We demonstrated that the loss of membrane integrity reflected in the elevated abundance of membrane proteins had a higher impact on seed aging progress than the MetO/Msr system. Proteome analyses enabled us to propose protein Sec61 and glyceraldehyde-3-phosphate dehydrogenase as potential longevity modulators in beech seeds.
Assuntos
Fagus , Metionina , Proteínas de Plantas , Proteômica , Sementes , Fagus/metabolismo , Metionina/metabolismo , Metionina/análogos & derivados , Sementes/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Germinação , Espécies Reativas de Oxigênio/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Core facilities have a different mission than academic research labs. Accordingly, they require different career paths and structures.
RESUMO
Metacaspases are cysteine proteases present in plants, fungi and protists. While the association of metacaspases with cell death is studied in a range of organisms, their native substrates are largely unknown. Here, we explored the in vivo proteolytic landscape of the two metacaspases, CrMCA-I and CrMCA-II, present in the green freshwater alga Chlamydomonas reinhardtii, using mass spectrometry-based degradomics approach, during control conditions and salt stress. Comparison between the cleavage events of CrMCA-I and CrMCA-II in metacaspase mutants revealed unique cleavage preferences and substrate specificity. Degradome analysis demonstrated the relevance of the predicted metacaspase substrates to the physiology of C. reinhardtii cells and its adaptation during salt stress. Functional enrichment analysis indicated an involvement of CrMCA-I in the catabolism of carboxylic acids, while CrMCA-II plays an important role in photosynthesis and translation. Altogether, our findings suggest distinct cellular functions of the two metacaspases in C. reinhardtii during salt stress response.
Assuntos
Chlamydomonas reinhardtii , Proteólise , Estresse Salino , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/metabolismo , Proteólise/efeitos dos fármacos , Caspases/metabolismo , Caspases/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMO
Human respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Nonstructural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. Likely, NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of the NS1 interaction partners, we performed three complementary protein-protein interaction screens, i.e., BioID, MAPPIT, and KISS. To closely mimic a natural infection, the BioID proximity screen was performed using a recombinant RSV in which the NS1 protein is fused to a biotin ligase. Remarkably, MED25, a subunit of the Mediator complex, was identified in all three performed screening methods as a potential NS1-interacting protein. We confirmed the interaction between MED25 and RSV NS1 by coimmunoprecipitation, not only upon overexpression of NS1 but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV can be enhanced in MED25 knockout A549 cells, suggesting a potential antiviral role of MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection. IMPORTANCE Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. A unique RSV protein, NS1, is largely responsible for this effect, probably through interaction with multiple host proteins. A better understanding of the interactions that occur between RSV NS1 and host proteins may help to identify targets for an effective antiviral therapy. We addressed this question by performing three complementary protein-protein interaction screens and identified MED25 as an RSV NS1-interacting protein. We propose a role in innate anti-RSV defense for this Mediator complex subunit.
Assuntos
Complexo Mediador , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Proteínas não Estruturais Virais , Células A549 , Humanos , Interferons/metabolismo , Complexo Mediador/genética , Complexo Mediador/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.
Assuntos
Biotina/química , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas , Arabidopsis/citologia , Arabidopsis/metabolismo , Biotina/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lotus/genética , Lotus/metabolismo , Solanum lycopersicum/química , Solanum lycopersicum/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Temperatura , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Nicotiana/metabolismoRESUMO
[Figure: see text].
Assuntos
Cardiopatias Congênitas/genética , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Processamento de Proteína Pós-Traducional , Acetilação , Células Cultivadas , Criança , Haploinsuficiência , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação de Sentido Incorreto , Proteoma/metabolismoRESUMO
Proteomics research infrastructures and core facilities within the Core for Life alliance advocate for community policies for quality control to ensure high standards in proteomics services.
Assuntos
Proteômica , Espectrometria de MassasRESUMO
BACKGROUND: It is increasingly recognized that conventional food production systems are not able to meet the globally increasing protein needs, resulting in overexploitation and depletion of resources, and environmental degradation. In this context, microbial biomass has emerged as a promising sustainable protein alternative. Nevertheless, often no consideration is given on the fact that the cultivation conditions affect the composition of microbial cells, and hence their quality and nutritional value. Apart from the properties and nutritional quality of the produced microbial food (ingredient), this can also impact its sustainability. To qualitatively assess these aspects, here, we investigated the link between substrate availability, growth rate, cell composition and size of Cupriavidus necator and Komagataella phaffii. RESULTS: Biomass with decreased nucleic acid and increased protein content was produced at low growth rates. Conversely, high rates resulted in larger cells, which could enable more efficient biomass harvesting. The proteome allocation varied across the different growth rates, with more ribosomal proteins at higher rates, which could potentially affect the techno-functional properties of the biomass. Considering the distinct amino acid profiles established for the different cellular components, variations in their abundance impacts the product quality leading to higher cysteine and phenylalanine content at low growth rates. Therefore, we hint that costly external amino acid supplementations that are often required to meet the nutritional needs could be avoided by carefully applying conditions that enable targeted growth rates. CONCLUSION: In summary, we demonstrate tradeoffs between nutritional quality and production rate, and we discuss the microbial biomass properties that vary according to the growth conditions.