RESUMO
The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.
Assuntos
Nicotinamida-Nucleotídeo Adenililtransferase , Pró-Fármacos , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Microscopia Crioeletrônica , Células HEK293 , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Pró-Fármacos/metabolismo , Ligação Proteica , Especificidade por Substrato , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP-dependent peri-centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP-tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP-tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de-clustering, prolonged multipolar mitosis, and cell death. 3D-organotypic invasion assays reveal that CCB02 has broad anti-invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)-resistant EGFR-mutant non-small-cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug-resistant cancers exhibiting high incidence of centrosome amplification.
Assuntos
Centrossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/administração & dosagem , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HeLa , Humanos , Camundongos , Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ABCA3 (ATP-binding cassette subfamily A member 3) is a lipid transporter expressed in alveolar type II cells and localized in the limiting membrane of lamellar bodies. It is crucial for pulmonary surfactant storage and homeostasis. Mutations in the ABCA3 gene are the most common genetic cause of respiratory distress syndrome in mature newborns and of interstitial lung disease in children. Apart from lung transplant, there is no cure available. To address the lack of causal therapeutic options for ABCA3 deficiency, a rapid and reliable approach is needed to investigate variant-specific molecular mechanisms and to identify pharmacologic modulators for monotherapies or combination therapies. To this end, we developed a phenotypic cell-based assay to autonomously identify ABCA3 wild-type-like or mutant-like cells by using machine learning algorithms aimed at identifying morphologic differences in wild-type and mutant cells. The assay was subsequently used to identify new drug candidates for ABCA3-specific molecular correction by using high-content screening of 1,280 Food and Drug Administration-approved small molecules. Cyclosporin A was identified as a potent corrector, specific for some but not all ABCA3 variants. Results were validated by using our previously established functional small-format assays. Hence, cyclosporin A may be selected for orphan drug evaluation in controlled repurposing trials in patients.
Assuntos
Doenças Pulmonares Intersticiais , Surfactantes Pulmonares , Síndrome do Desconforto Respiratório do Recém-Nascido , Transportadores de Cassetes de Ligação de ATP/genética , Criança , Ciclosporina/farmacologia , Humanos , Recém-Nascido , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/genética , Mutação/genética , Síndrome do Desconforto Respiratório do Recém-Nascido/genéticaRESUMO
The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N3-methylcytosine (m3C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N1-methyladenosine (m1A) in tRNA or formation of 5-formyl cytosine (f5C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPRmt) in C. elegans.
Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Mitocôndrias/metabolismo , RNA Mitocondrial/metabolismo , Células A549 , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Animais , Caenorhabditis elegans , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Células HT29 , Células HeLa , Humanos , Camundongos , Microscopia Eletrônica , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio/fisiologia , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
Epithelial cell-adhesion molecule (EpCAM) is a transmembrane protein that regulates cell cycle progression and differentiation and is overexpressed in many carcinomas. The EpCAM-induced mitogenic cascade is activated via regulated intramembrane proteolysis (RIP) of EpCAM by ADAM and γ-secretases, generating the signaling-active intracellular domain EpICD. Because of its expression pattern and molecular function, EpCAM is a valuable target in prognostic and therapeutic approaches for various carcinomas. So far, several immunotherapeutic strategies have targeted the extracellular domain of EpCAM. However, targeting the intracellular signaling cascade of EpCAM holds promise for specifically interfering with EpCAM's proliferation-stimulating signaling cascade. Here, using a yellow fluorescence protein-tagged version of the C-terminal fragment of EpCAM, we established a high-content screening (HCS) of a small-molecule compound library (n = 27,280) and characterized validated hits that target EpCAM signaling. In total, 128 potential inhibitors were initially identified, of which one compound with robust inhibitory effects on RIP of EpCAM was analyzed in greater detail. In summary, our study demonstrates that the development of an HCS for small-molecule inhibitors of the EpCAM signaling pathway is feasible. We propose that this approach may also be useful for identifying chemical compounds targeting other disorders involving membrane cleavage-dependent signaling pathways.
Assuntos
Molécula de Adesão da Célula Epitelial/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Molécula de Adesão da Célula Epitelial/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Bibliotecas de Moléculas Pequenas/química , Transcrição Gênica/efeitos dos fármacosRESUMO
Constitutive NF-κB signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-κB pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys63-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6-Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-κB activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6-Ubc13 interaction and thereby counteract NF-κB signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6-Ubc13 interaction that reduces TRAF6-Ubc13 activity both in vitro and in cells. We found that this compound, C25-140, impedes NF-κB activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical in vivo mouse models. Hence, the first-in-class TRAF6-Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Inflamação/tratamento farmacológico , Psoríase/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mapas de Interação de Proteínas , Psoríase/metabolismo , Psoríase/patologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/antagonistas & inibidoresRESUMO
The ubiquitin (Ub)-related modifier Urm1 functions as a sulfur carrier in tRNA thiolation by means of a mechanism that requires the formation of a thiocarboxylate at the C-terminal glycine residue of Urm1. However, whether Urm1 plays an additional role as a Ub-like protein modifier remains unclear. Here, we show that Urm1 is conjugated to lysine residues of target proteins and that oxidative stress enhances protein urmylation in both Saccharomyces cerevisiae and mammalian cells. Similar to ubiquitylation, urmylation involves a thioester intermediate and results in the formation of a covalent peptide bond between Urm1 and its substrates. In contrast to modification by canonical Ub-like modifiers, however, conjugation of Urm1 involves a C-terminal thiocarboxylate of the modifier. We have confirmed that the peroxiredoxin Ahp1 is such a substrate in S. cerevisiae and found that Urm1 targets a specific lysine residue of Ahp1 in vivo. In addition, we have identified several unique substrates in mammalian cells and show that Urm1 targets at least two pathways on oxidant treatment. First, Urm1 is appended to lysine residues of three components that function in its own pathway (i.e., MOCS3, ATPBD3, and CTU2). Second, Urm1 is conjugated to the nucleocytoplasmic shuttling factor cellular apoptosis susceptibility protein. Thus, Urm1 has a conserved dual role by integrating the functions of prokaryotic sulfur carriers with those of eukaryotic protein modifiers of the Ub family.
Assuntos
Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquitinas/fisiologia , Cromatografia Líquida , Diamida/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Estresse Oxidativo , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
Cell death, such as apoptosis and ferroptosis, play essential roles in the process of development, homeostasis, and pathogenesis of acute and chronic diseases. The increasing number of studies investigating cell death types in various diseases, particularly cancer and degenerative diseases, has raised hopes for their modulation in disease therapies. However, identifying the presence of a particular cell death type is not an obvious task, as it requires computationally intensive work and costly experimental assays. To address this challenge, we present CellDeathPred, a novel deep-learning framework that uses high-content imaging based on cell painting to distinguish cells undergoing ferroptosis or apoptosis from healthy cells. In particular, we incorporate a deep neural network that effectively embeds microscopic images into a representative and discriminative latent space, classifies the learned embedding into cell death modalities, and optimizes the whole learning using the supervised contrastive loss function. We assessed the efficacy of the proposed framework using cell painting microscopy data sets from human HT-1080 cells, where multiple inducers of ferroptosis and apoptosis were used to trigger cell death. Our model confidently separates ferroptotic and apoptotic cells from healthy controls, with an average accuracy of 95% on non-confocal data sets, supporting the capacity of the CellDeathPred framework for cell death discovery.
RESUMO
Ferroptosis is a regulated cell death modality that occurs upon iron-dependent lipid peroxidation. Recent research has identified many regulators that induce or inhibit ferroptosis; yet, many regulatory processes and networks remain to be elucidated. In this study, we performed a chemical genetics screen using small molecules with known mode of action and identified two agonists of the nuclear receptor Farnesoid X Receptor (FXR) that suppress ferroptosis, but not apoptosis or necroptosis. We demonstrate that in liver cells with high FXR levels, knockout or inhibition of FXR sensitized cells to ferroptotic cell death, whereas activation of FXR by bile acids inhibited ferroptosis. Furthermore, FXR inhibited ferroptosis in ex vivo mouse hepatocytes and human hepatocytes differentiated from induced pluripotent stem cells. Activation of FXR significantly reduced lipid peroxidation by upregulating the ferroptosis gatekeepers GPX4, FSP1, PPARα, SCD1, and ACSL3. Together, we report that FXR coordinates the expression of ferroptosis-inhibitory regulators to reduce lipid peroxidation, thereby acting as a guardian of ferroptosis.
Assuntos
Ácidos e Sais Biliares , Ferroptose , Animais , Humanos , Camundongos , Ácidos e Sais Biliares/metabolismo , Hepatócitos/metabolismo , Peroxidação de Lipídeos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Protein ubiquitination is an essential post-translational modification that regulates a large number of cellular processes. This reaction is facilitated by the consecutive action of three central enzymes, i.e., E1 activating enzyme, E2 conjugating enzyme, and the E3 ligase. More than 600 E3 enzymes guarantee the specificity and selectivity of these reactions and thus represent an exciting, while highly underrepresented, class of drug targets. Specific methods can be employed to monitor their activity and thus query compound libraries for inhibitory small molecules. Here, we describe two protocols-one high-throughput and one low-throughput method-to detect E3 ligase activity and test small molecule modulation. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: AlphaScreen assay to measure TRAF6-Ubc13 interaction Basic Protocol 2: Gel-based in vitro ubiquitination assay (K63-linked chains).
Assuntos
Fator 6 Associado a Receptor de TNF , Ubiquitina-Proteína Ligases , Processamento de Proteína Pós-Traducional , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Determining cell death mechanisms occurring in patient and animal tissues is a longstanding goal that requires suitable biomarkers and accurate quantification. However, effective methods remain elusive. To develop more powerful and unbiased analytic frameworks, we developed a machine learning approach for automated cell death classification. Image sets were collected of HT-1080 fibrosarcoma cells undergoing ferroptosis or apoptosis and stained with an anti-transferrin receptor 1 (TfR1) antibody, together with nuclear and F-actin staining. Features were extracted using high-content-analysis software, and a classifier was constructed by fitting a multinomial logistic lasso regression model to the data. The prediction accuracy of the classifier within three classes (control, ferroptosis, apoptosis) was 93%. Thus, TfR1 staining, combined with nuclear and F-actin staining, can reliably detect both apoptotic and ferroptotis cells when cell features are analyzed in an unbiased manner using machine learning, providing a method for unbiased analysis of modes of cell death.
Assuntos
Ferroptose , Receptores da Transferrina , Actinas , Apoptose , Biomarcadores , Humanos , Aprendizado de MáquinaRESUMO
Complex mixtures containing natural products are still an interesting source of novel drug candidates. High content screening (HCS) is a popular tool to screen for such. In particular, multiplexed HCS assays promise comprehensive bioactivity profiles, but generate also high amounts of data. Yet, only some machine learning (ML) applications for data analysis are available and these usually require a profound knowledge of the underlying cell biology. Unfortunately, there are no applications that simply predict if samples are biologically active or not (any kind of bioactivity). Within this work, we benchmark ML algorithms for binary classification, starting with classical ML models, which are the standard classifiers of the scikit-learn library or ensemble models of these classifiers (a total of 92 models tested). Followed by a partial least square regression (PLSR)-based classification (44 tested models in total) and simple artificial neural networks (ANNs) with dense layers (72 tested models in total). In addition, a novelty detection (ND) was examined, which is supposed to handle unknown patterns. For the final analysis the models, with and without upstream ND, were tested with two independent data sets. In our analysis, a stacking model, an ensamble model of class ML algorithms, performed best to predict new and unknown data. ND improved the predictions of the models and was useful to handle unknown patterns. Importantly, the classifier presented here can be easily rebuilt and be adapted to the data and demands of other groups. The hit detector (ND + stacking model) is universal and suitable for a broader application to support the search for new drug candidates.
RESUMO
Trypanosomiases are life-threatening infections of humans and livestock, and novel effective therapeutic approaches are needed. Trypanosoma compartmentalize glycolysis into specialized organelles termed glycosomes. Most of the trypanosomal glycolytic enzymes harbor a peroxisomal targeting signal-1 (PTS1) which is recognized by the soluble receptor PEX5 to facilitate docking and translocation of the cargo into the glycosomal lumen. Given its pivotal role in the glycosomal protein import, the PEX5-PTS1 interaction represents a potential target to inhibit import of glycolytic enzymes and thus kill the parasite. We developed a fluorescence polarization (FP)-based assay for monitoring the PEX5-PTS1 interaction and performed a High Throughput Screening (HTS) campaign to identify small molecule inhibitors of the interaction. Six of the identified hits passed orthogonal selection criteria and were found to inhibit parasite growth in cell culture. Our results validate PEX5 as a target for small molecule inhibitors and provide scaffolds suitable for further pre-clinical development of novel trypanocidal compounds.
Assuntos
Receptores Citoplasmáticos e Nucleares , Trypanosoma , Proteínas de Transporte/metabolismo , Humanos , Microcorpos/metabolismo , Receptor 2 de Sinal de Orientação para Peroxissomos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Peroxissomos/metabolismo , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Trypanosoma/metabolismoRESUMO
Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic stage of the disease, urgently calling for the identification of new targets and drug candidates. Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzo[b,f][1,4]thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases.
Assuntos
Tripanossomicidas , Trypanosoma brucei brucei , Trypanosoma , Proteínas de Membrana , Microcorpos , Transporte Proteico/fisiologia , Relação Estrutura-Atividade , Tripanossomicidas/farmacologiaRESUMO
The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Acriflavina , Animais , Antivirais/química , Antivirais/farmacologia , Humanos , Camundongos , Simulação de Acoplamento Molecular , PandemiasRESUMO
Protein-protein interactions (PPI) are involved in a myriad of cellular processes, and their deregulation can lead to many diseases. One such process is protein ubiquitination that requires an orchestrated action of three key enzymes to add ubiquitin moieties to substrate proteins. Importantly, this process is reversible through deubiquitinating enzymes. Both ubiquitination and deubiquitination require many PPIs that once classified can be utilized to identify small molecule inhibitors counteracting these reactions. Here, we study the protein-protein interaction between the two deubiquitinating enzymes OTUB1 and OTUD6B and report for the first time that both proteins directly interact with each other. We describe the GFP-Trap immunoprecipitation as a cell-based method to analyze the OTUD6B-OTUB1 interaction in the cellular context and the AlphaScreen (amplified luminescent proximity homogeneous assay) assay as a tool to detect direct interactions and to search for PPI inhibitors.
Assuntos
Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Ensaios de Triagem em Larga Escala , Imunoprecipitação , Mapas de Interação de Proteínas , Proteômica , Cisteína Endopeptidases/genética , Enzimas Desubiquitinantes , Endopeptidases/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , UbiquitinaçãoRESUMO
Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such '2-cell-like-cells' (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Totipotentes/citologia , Tretinoína/fisiologia , Acitretina/farmacologia , Animais , Massa Celular Interna do Blastocisto/citologia , Diferenciação Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Redes Reguladoras de Genes/genética , Genes Reporter , Isotretinoína/farmacologia , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Piperazinas/farmacologia , Pirazóis/farmacologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , RNA-Seq , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco Totipotentes/efeitos dos fármacos , Transcrição Gênica , Tretinoína/antagonistas & inibidores , Tretinoína/farmacologia , Receptor gama de Ácido RetinoicoRESUMO
BACKGROUND AND PURPOSE: Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/ß-catenin signalling is reduced in emphysema, and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/ß-catenin signalling and assess its potential to activate lung epithelial cells and repair. EXPERIMENTAL APPROACH: We screened 1216 human-approved compounds for Wnt/ß-catenin signalling activation using luciferase reporter cells and selected candidates based on their computationally predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell-derived lung organoids and in vivo using a murine elastase-induced emphysema model. KEY RESULTS: The primary screen identified 16 compounds that significantly induced Wnt/ß-catenin-dependent luciferase activity. Selected compounds activated Wnt/ß-catenin signalling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/ß-catenin inhibition, confirming the Wnt/ß-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation, and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces pro-regenerative signalling in emphysema. CONCLUSION AND IMPLICATIONS: Using a drug screen based on Wnt/ß-catenin activity, organoid assays and a murine emphysema model, amlexanox was identified as a novel potential therapeutic agent for emphysema.
Assuntos
Preparações Farmacêuticas , beta Catenina , Aminopiridinas , Animais , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organoides , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
BACKGROUND: Studies suggest that general anesthetics like isoflurane and sevoflurane may aggravate Alzheimer's disease (AD) neuropathogenesis, e.g., increased amyloid-ß (Aß) protein aggregation resulting in synaptotoxicity and cognitive dysfunction. Other studies showed neuroprotective effects, e.g., with xenon. OBJECTIVE: In the present study, we want to detail the interactions of inhalational anesthetics with Aß-derived pathology. We hypothesize xenon-mediated beneficial mechanisms regarding Aß oligomerization and Aß-mediated neurotoxicity on processes related to cognition. METHODS: Oligomerization of Aß1-42 in the presence of anesthetics has been analyzed by means of TR-FRET and silver staining. For monitoring changes in neuronal plasticity due to anesthetics and Aß1-42, Aß1-40, pyroglutamate-modified amyloid-(AßpE3), and nitrated Aß (3NTyrAß), we quantified long-term potentiation (LTP) and spine density. We analyzed network activity in the hippocampus via voltage-sensitive dye imaging (VSDI) and cognitive performance and Aß plaque burden in transgenic AD mice (ArcAß) after anesthesia. RESULTS: Whereas isoflurane and sevoflurane did not affect Aß1-42 aggregation, xenon alleviated the propensity for aggregation and partially reversed AßpE3 induced synaptotoxic effects on LTP. Xenon and sevoflurane reversed Aß1-42-induced spine density attenuation. In the presence of Aß1-40 and AßpE3, anesthetic-induced depression of VSDI-monitored signaling recovered after xenon, but not isoflurane and sevoflurane removal. In slices pretreated with Aß1-42 or 3NTyrAß, activity did not recover after washout. Cognitive performance and plaque burden were unaffected after anesthetizing WT and ArcAß mice. CONCLUSION: None of the anesthetics aggravated Aß-derived AD pathology in vivo. However, Aß and anesthetics affected neuronal activity in vitro, whereby xenon showed beneficial effects on Aß1-42 aggregation, LTP, and spine density.
Assuntos
Doença de Alzheimer/fisiopatologia , Anestésicos Inalatórios/administração & dosagem , Isoflurano/administração & dosagem , Placa Amiloide/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Hipocampo/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Xenônio/administração & dosagemRESUMO
The biological function and disease association of human endogenous retroviruses (HERVs) are largely elusive. HERV-K(HML-2) has been associated with neurotoxicity, but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation using CRISPR engineering to activate or repress its expression levels in a human-pluripotent-stem-cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell-type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell-type-specific role for HERV-K(HML-2) in cortical neuron development.