RESUMO
Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an α4ß4 architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4-diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.
Assuntos
Proteínas de Bactérias , Betaproteobacteria , Ácidos Graxos Dessaturases , Estigmasterol , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Molibdênio/química , Estigmasterol/metabolismo , Betaproteobacteria/enzimologia , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Hidroxilação/genética , Flavinas/metabolismoRESUMO
Transient stress experiences not only trigger acute stress responses, but can also have long-lasting effects on cellular functions. In Caenorhabditis elegans, a brief exposure to heat shock during early adulthood extends lifespan and improves stress resistance, a phenomenon known as heat hormesis. Here, we investigated the prolonged effect of hormetic heat stress on the transcriptome of worms and found that the canonical heat shock response is followed by a profound transcriptional reprogramming in the post-stress period. This reprogramming relies on the endoribonuclease ENDU-2 but not the heat shock factor 1. ENDU-2 co-localizes with chromatin and interacts with RNA polymerase II, enabling specific regulation of transcription after the stress period. Failure to activate the post-stress response does not affect the resistance of animals to heat shock but eliminates the beneficial effects of hormetic heat stress. In summary, our work discovers that the RNA-binding protein ENDU-2 mediates the long-term impacts of transient heat stress via reprogramming transcriptome after stress exposure.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Transcriptoma , Hormese/fisiologia , Resposta ao Choque Térmico/genética , Longevidade/fisiologiaRESUMO
Phosphorylation-dependent signal transduction plays an important role in regulating the functions and fate of skeletal muscle cells. Central players in the phospho-signaling network are the protein kinases AKT, S6K, and RSK as part of the PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK pathways. However, despite their functional importance, knowledge about their specific targets is incomplete because these kinases share the same basophilic substrate motif RxRxxp[ST]. To address this, we performed a multifaceted quantitative phosphoproteomics study of skeletal myotubes following kinase inhibition. Our data corroborate a cross talk between AKT and RAF, a negative feedback loop of RSK on ERK, and a putative connection between RSK and PI3K signaling. Altogether, we report a kinase target landscape containing 49 so far unknown target sites. AKT, S6K, and RSK phosphorylate numerous proteins involved in muscle development, integrity, and functions, and signaling converges on factors that are central for the skeletal muscle cytoskeleton. Whereas AKT controls insulin signaling and impinges on GTPase signaling, nuclear signaling is characteristic for RSK. Our data further support a role of RSK in glucose metabolism. Shared targets have functions in RNA maturation, stability, and translation, which suggests that these basophilic kinases establish an intricate signaling network to orchestrate and regulate processes involved in translation.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Fibras Musculares Esqueléticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa , Proteínas Quinases S6 Ribossômicas 70-kDaRESUMO
The analysis of data collected using design of experiments (DoE) is the current gold standard to determine the influence of input parameters and their interactions on process performance and product quality. In early development, knowledge on the bioprocess of a new product is limited. Many input parameters need to be investigated for a thorough investigation. For eukaryotic cell cultures, intensified DoE (iDoE) has been proposed as efficient tool, requiring fewer bioreactor runs by introducing setpoint changes during the bioprocess. We report the first successful application of iDoE to mammalian cell culture, performing sequential setpoint changes in the growth phase for the selected input parameters temperature and dissolved oxygen. The process performance data were analyzed using ordinary least squares regression. Our results indicate iDoE to be applicable to mammalian bioprocesses and to be a cost-efficient option to inform modeling early on during process development. Even though only half the number of bioreactor runs were used in comparison to a classical DoE approach, the resulting models revealed comparable input-output relations. Being able to examine several setpoint levels within one bioreactor run, we confirm iDoE to be a promising tool to speed up biopharmaceutical process development.
RESUMO
Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human mitochondrial proteome. We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context.
Assuntos
Mitocôndrias , Proteoma , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteoma/metabolismoRESUMO
The calculation of temporally varying upstream process outcomes is a challenging task. Over the last years, several parametric, semi-parametric as well as non-parametric approaches were developed to provide reliable estimates for key process parameters. We present generic and product-specific recurrent neural network (RNN) models for the computation and study of growth and metabolite-related upstream process parameters as well as their temporal evolution. Our approach can be used for the control and study of single product-specific large-scale manufacturing runs as well as generic small-scale evaluations for combined processes and products at development stage. The computational results for the product titer as well as various major upstream outcomes in addition to relevant process parameters show a high degree of accuracy when compared to experimental data and, accordingly, a reasonable predictive capability of the RNN models. The calculated values for the root-mean squared errors of prediction are significantly smaller than the experimental standard deviation for the considered process run ensembles, which highlights the broad applicability of our approach. As a specific benefit for platform processes, the generic RNN model is also used to simulate process outcomes for different temperatures in good agreement with experimental results. The high level of accuracy and the straightforward usage of the approach without sophisticated parameterization and recalibration procedures highlight the benefits of the RNN models, which can be regarded as promising alternatives to existing parametric and semi-parametric methods.
RESUMO
The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig-like domain 20 of FLNc, is doubly phosphorylated. The protein kinases responsible for this dual-site phosphorylation are Akt and PKCα. Proximity proteomics and interaction analysis identified filamin A-interacting protein 1 (FILIP1) as direct FLNc binding partner. FILIP1 binding induces filamin degradation, thereby negatively regulating its function. Here, dual-site phosphorylation of FLNc not only reduces FILIP1 binding, providing a mechanism to shield FLNc from FILIP1-mediated degradation, but also enables fast dynamics of FLNc necessary for its function as signaling adaptor in cross-striated muscle cells.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Filaminas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Motivos de Aminoácidos , Células HEK293 , Humanos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Proteólise , Proteoma/análise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
The generation of reactive oxygen species (ROS) is inevitably linked to life. However, the precise role of ROS in signalling and specific targets is largely unknown. We perform a global proteomic analysis to delineate the yeast redoxome to a depth of more than 4,300 unique cysteine residues in over 2,200 proteins. Mapping of redox-active thiols in proteins exposed to exogenous or endogenous mitochondria-derived oxidative stress reveals ROS-sensitive sites in several components of the translation apparatus. Mitochondria are the major source of cellular ROS. We demonstrate that increased levels of intracellular ROS caused by dysfunctional mitochondria serve as a signal to attenuate global protein synthesis. Hence, we propose a universal mechanism that controls protein synthesis by inducing reversible changes in the translation machinery upon modulating the redox status of proteins involved in translation. This crosstalk between mitochondria and protein synthesis may have an important contribution to pathologies caused by dysfunctional mitochondria.
Assuntos
Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Oxirredução , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Compostos de Sulfidrila/químicaRESUMO
Twin-arginine translocation (Tat) systems transport folded proteins across cellular membranes with the concerted action of mostly three membrane proteins: TatA, TatB, and TatC. Hetero-oligomers of TatB and TatC form circular substrate-receptor complexes with a central binding cavity for twin-arginine-containing signal peptides. After binding of the substrate, energy from an electro-chemical proton gradient is transduced into the recruitment of TatA oligomers and into the actual translocation event. We previously reported that Tat-dependent protein translocation into membrane vesicles of Escherichia coli is blocked by the compound N,N'-dicyclohexylcarbodiimide (DCCD, DCC). We have now identified a highly conserved glutamate residue in the transmembrane region of E. coli TatC, which when modified by DCCD interferes with the deep insertion of a Tat signal peptide into the TatBC receptor complex. Our findings are consistent with a hydrophobic binding cavity formed by TatB and TatC inside the lipid bilayer. Moreover, we found that DCCD mediates discrete intramolecular cross-links of E. coli TatC involving both its N- and C-tails. These results confirm the close proximity of two distant sequence sections of TatC proposed to concertedly function as the primary docking site for twin-arginine signal peptides.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Arginina/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Dicicloexilcarbodi-Imida/farmacologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana Transportadoras/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Sinais Direcionadores de Proteínas/fisiologia , Especificidade por SubstratoRESUMO
Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles.
Assuntos
Espectrometria de Massas/métodos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Técnicas de Silenciamento de Genes , Microcorpos/metabolismo , Membranas Mitocondriais/metabolismo , Transporte Proteico , Especificidade por SubstratoRESUMO
Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5'ppp-dsRNA sensing and virtually abrogate RIG-I activation.
Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , RNA Interferente Pequeno/genética , Receptores do Ácido Retinoico/metabolismo , Células A549 , Animais , Células Cultivadas , Retroalimentação Fisiológica , Células HEK293 , Humanos , Camundongos , Fosforilação , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Genética , Hepacivirus/genética , MicroRNAs/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Linhagem Celular Tumoral , Resistência à Doença/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Loci Gênicos , Genoma Humano , Células HEK293 , Hepatócitos/metabolismo , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , MicroRNAs/metabolismo , RNA Interferente Pequeno/metabolismo , Análise de Sequência de DNA , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Replicação Viral/genéticaRESUMO
The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling.
Assuntos
Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Proteína Quinase C/metabolismo , Proteômica/métodos , Sarcômeros/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Estimulação Elétrica , Filaminas/metabolismo , Camundongos , Mioblastos/metabolismo , Fosforilação , Mapas de Interação de ProteínasRESUMO
Tumor relapse is associated with dismal prognosis, but responsible biological principles remain incompletely understood. To isolate and characterize relapse-inducing cells, we used genetic engineering and proliferation-sensitive dyes in patient-derived xenografts of acute lymphoblastic leukemia (ALL). We identified a rare subpopulation that resembled relapse-inducing cells with combined properties of long-term dormancy, treatment resistance, and stemness. Single-cell and bulk expression profiling revealed their similarity to primary ALL cells isolated from pediatric and adult patients at minimal residual disease (MRD). Therapeutically adverse characteristics were reversible, as resistant, dormant cells became sensitive to treatment and started proliferating when dissociated from the in vivo environment. Our data suggest that ALL patients might profit from therapeutic strategies that release MRD cells from the niche.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica/métodos , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Análise de Sequência de RNA/métodos , Adulto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proliferação de Células , Criança , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Recidiva Local de Neoplasia/genética , Transplante de Neoplasias , Neoplasia Residual/genética , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prognóstico , Análise de Célula Única , Células Tumorais CultivadasRESUMO
Hepatitis B virus (HBV) is a promising target for therapies based on RNA interference (RNAi) since it replicates via RNA transcripts that are vulnerable to RNAi silencing. Clinical translation of RNAi technology, however, requires improvements in potency, specificity and safety. To this end, we systematically compared different strategies to express anti-HBV short hairpin RNA (shRNA) in a pre-clinical immunocompetent hepatitis B mouse model. Using recombinant Adeno-associated virus (AAV) 8 vectors for delivery, we either (i) embedded the shRNA in an artificial mi(cro)RNA under a liver-specific promoter; (ii) co-expressed Argonaute-2, a rate-limiting cellular factor whose saturation with excess RNAi triggers can be toxic; or (iii) co-delivered a decoy ("TuD") directed against the shRNA sense strand to curb off-target gene regulation. Remarkably, all three strategies minimised adverse side effects as compared to a conventional shRNA vector that caused weight loss, liver damage and dysregulation of > 100 hepatic genes. Importantly, the novel AAV8 vector co-expressing anti-HBV shRNA and TuD outperformed all other strategies regarding efficiency and persistence of HBV knock-down, thus showing substantial promise for clinical translation.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/terapia , RNA Interferente Pequeno/farmacologia , Animais , Antivirais/efeitos adversos , Antivirais/uso terapêutico , Dependovirus/genética , Modelos Animais de Doenças , Portadores de Fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Expressão Gênica , Vetores Genéticos , Camundongos , Transdução GenéticaAssuntos
Proteínas Sanguíneas/metabolismo , Dermatite Atópica/sangue , Adolescente , Adulto , Biomarcadores/sangue , Proteínas Sanguíneas/imunologia , Citocinas/sangue , Dermatite Atópica/imunologia , Feminino , Humanos , Análise dos Mínimos Quadrados , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Modelos Estatísticos , Índice de Gravidade de Doença , Adulto JovemRESUMO
Naive CD4(+) T cells are the common precursors of multiple effector and memory T-cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell-like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4(+) T cells and their changes during the early phase of T-cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation technology was applied with subsequent quantitative liquid chromatography-tandem MS to generate a data set describing the surface proteome of primary human naive CD4(+) T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic data set and to analyze the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous data set, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4(+) T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation, and predicted subcellular localization, and correlated the proteomics result with this transcriptional data set. This extensive surface atlas provides an overall naive CD4(+) T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T-cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Ativação Linfocitária/imunologia , Proteômica/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Membrana Celular/metabolismo , Análise por Conglomerados , Simulação por Computador , Citometria de Fluxo , Perfilação da Expressão Gênica , Ontologia Genética , Glicoproteínas/metabolismo , Humanos , Proteoma/metabolismo , Reprodutibilidade dos Testes , Transcriptoma/genéticaRESUMO
UNLABELLED: With the widespread availability of high-throughput experimental technologies it has become possible to study hundreds to thousands of cellular factors simultaneously, such as coding- or non-coding mRNA or protein concentrations. Still, extracting information about the underlying regulatory or signaling interactions from these data remains a difficult challenge. We present a flexible approach towards network inference based on linear programming. Our method reconstructs the interactions of factors from a combination of perturbation/non-perturbation and steady-state/time-series data. We show both on simulated and real data that our methods are able to reconstruct the underlying networks fast and efficiently, thus shedding new light on biological processes and, in particular, into disease's mechanisms of action. We have implemented the approach as an R package available through bioconductor. AVAILABILITY AND IMPLEMENTATION: This R package is freely available under the Gnu Public License (GPL-3) from bioconductor.org (http://bioconductor.org/packages/release/bioc/html/lpNet.html) and is compatible with most operating systems (Windows, Linux, Mac OS) and hardware architectures. CONTACT: bettina.knapp@helmholtz-muenchen.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Redes e Vias Metabólicas , Programação Linear , Transdução de Sinais , Software , Gráficos por Computador , Genoma Humano , Genômica , Humanos , Integração de SistemasRESUMO
The mechanical stability of epithelial cells, which protect organisms from harmful external factors, is maintained by hemidesmosomes via the interaction between plectin 1a (P1a) and integrin α6ß4. Binding of calcium-calmodulin (Ca(2+)-CaM) to P1a together with phosphorylation of integrin ß4 disrupts this complex, resulting in disassembly of hemidesmosomes. We present structures of the P1a actin binding domain either in complex with the N-ter lobe of Ca(2+)-CaM or with the first pair of integrin ß4 fibronectin domains. Ca(2+)-CaM binds to the N-ter isoform-specific tail of P1a in a unique manner, via its N-ter lobe in an extended conformation. Structural, cell biology, and biochemical studies suggest the following model: binding of Ca(2+)-CaM to an intrinsically disordered N-ter segment of plectin converts it to an α helix, which repositions calmodulin to displace integrin ß4 by steric repulsion. This model could serve as a blueprint for studies aimed at understanding how Ca(2+)-CaM or EF-hand motifs regulate F-actin-based cytoskeleton.