RESUMO
Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.
Assuntos
Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Mucosa Intestinal/patologia , Antígenos CD/metabolismo , Apirase/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Morte Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Criança , Estudos de Coortes , Colo/patologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Dipiridamol/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Homeostase/efeitos dos fármacos , Humanos , Imunoglobulina G/sangue , Memória Imunológica , Inflamação/patologia , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/genética , Interferon Tipo I/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metilprednisolona/farmacologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismoRESUMO
Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase ß and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase ß and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1-/- cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.
Assuntos
Reparo do DNA/genética , DNA/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Animais , Linhagem Celular , Quebras de DNA de Cadeia Simples , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Polimerase beta/metabolismo , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Camptotecina/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Células HeLa , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Inibidores da Topoisomerase I/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Quinase 1 Polo-LikeRESUMO
Immune responses need to be tightly controlled to avoid excessive inflammation and prevent unwanted host damage. Here we report that germinal center kinase MST4 responded dynamically to bacterial infection and acted as a negative regulator of inflammation. We found that MST4 directly interacted with and phosphorylated the adaptor TRAF6 to prevent its oligomerization and autoubiquitination. Accordingly, MST4 did not inhibit lipopolysaccharide-induced cytokine production in Traf6(-/-) embryonic fibroblasts transfected to express a mutant form of TRAF6 that cannot be phosphorylated at positions 463 and 486 (with substitution of alanine for threonine at those positions). Upon developing septic shock, mice in which MST4 was knocked down showed exacerbated inflammation and reduced survival, whereas heterozygous deletion of Traf6 (Traf6(+/-)) alleviated such deleterious effects. Our findings reveal a mechanism by which TRAF6 is regulated and highlight a role for MST4 in limiting inflammatory responses.
Assuntos
Inflamação/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Inflamação/induzido quimicamente , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Sepse/sangue , Choque Séptico/induzido quimicamente , Choque Séptico/metabolismoRESUMO
Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.
Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fibroblastos/efeitos dos fármacos , Interferon gama/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Quinase 8 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Fibroblastos/enzimologia , Fibroblastos/virologia , Células HCT116 , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , RNA Polimerase II/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Vesiculovirus/patogenicidadeRESUMO
Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point.
Assuntos
Proliferação de Células , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Células Epiteliais/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular , Mitógenos/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/enzimologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Camundongos , Modelos Biológicos , Fosforilação , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC.
Assuntos
Fibroblastos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Fibroblastos/enzimologia , Células HEK293 , Humanos , Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multienzimáticos , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Ferroptosis is a regulated necrosis process driven by iron-dependent lipid peroxidation. Although ferroptosis and cellular metabolism interplay with one another, whether mitochondria are involved in ferroptosis is under debate. Here, we demonstrate that mitochondria play a crucial role in cysteine-deprivation-induced ferroptosis but not in that induced by inhibiting glutathione peroxidase-4 (GPX4), the most downstream component of the ferroptosis pathway. Mechanistically, cysteine deprivation leads to mitochondrial membrane potential hyperpolarization and lipid peroxide accumulation. Inhibition of mitochondrial TCA cycle or electron transfer chain (ETC) mitigated mitochondrial membrane potential hyperpolarization, lipid peroxide accumulation, and ferroptosis. Blockage of glutaminolysis had the same inhibitory effect, which was counteracted by supplying downstream TCA cycle intermediates. Importantly, loss of function of fumarate hydratase, a tumor suppressor and TCA cycle component, confers resistance to cysteine-deprivation-induced ferroptosis. Collectively, this work demonstrates the crucial role of mitochondria in cysteine-deprivation-induced ferroptosis and implicates ferroptosis in tumor suppression.
Assuntos
Ciclo do Ácido Cítrico , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/enzimologia , Ferro/metabolismo , Peroxidação de Lipídeos , Mitocôndrias/enzimologia , Animais , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Glutamina/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/patologia , Mutação , Necrose , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
The health risks that arise from environmental exposures vary widely within and across human populations, and these differences are largely determined by genetic variation and gene-by-environment (gene-environment) interactions. However, risk assessment in laboratory mice typically involves isogenic strains and therefore, does not account for these known genetic effects. In this context, genetically heterogenous cell lines from laboratory mice are promising tools for population-based screening because they provide a way to introduce genetic variation in risk assessment without increasing animal use. Cell lines from genetic reference populations of laboratory mice offer genetic diversity, power for genetic mapping, and potentially, predictive value for in vivo experimentation in genetically matched individuals. To explore this further, we derived a panel of fibroblast lines from a genetic reference population of laboratory mice (the Diversity Outbred, DO). We then used high-content imaging to capture hundreds of cell morphology traits in cells exposed to the oxidative stress-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We employed dose-response modeling to capture latent parameters of response and we then used these parameters to identify several hundred cell morphology quantitative trait loci (cmQTL). Response cmQTL encompass genes with established associations with cellular responses to arsenic exposure, including Abcc4 and Txnrd1, as well as novel gene candidates like Xrcc2. Moreover, baseline trait cmQTL highlight the influence of natural variation on fundamental aspects of nuclear morphology. We show that the natural variants influencing response include both coding and non-coding variation, and that cmQTL haplotypes can be used to predict response in orthogonal cell lines. Our study sheds light on the major molecular initiating events of oxidative stress that are under genetic regulation, including the NRF2-mediated antioxidant response, cellular detoxification pathways, DNA damage repair response, and cell death trajectories.
Assuntos
Arsênio , Estresse Oxidativo , Locos de Características Quantitativas , Animais , Camundongos , Arsênio/toxicidade , Estresse Oxidativo/genética , Estresse Oxidativo/efeitos dos fármacos , Humanos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Linhagem Celular , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Interação Gene-Ambiente , Intoxicação por Arsênico/genética , Mapeamento CromossômicoRESUMO
Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.
Assuntos
Envelhecimento/fisiologia , Ácido Ascórbico/farmacologia , Diabetes Mellitus Experimental/prevenção & controle , Proteína do Retinoblastoma/metabolismo , Animais , Senescência Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Fator de Transcrição E2F1/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Fibroblastos/efeitos dos fármacos , Técnicas de Introdução de Genes , Células Secretoras de Insulina/patologia , Camundongos , Fosforilação , Gravidez , Proteína do Retinoblastoma/genética , Telômero/genéticaRESUMO
Aneuploidy, an incorrect chromosome number, is a hallmark of cancer. Compounds that cause lethality in aneuploid, but not euploid, cells could therefore provide new cancer therapies. We have identified the energy stress-inducing agent AICAR, the protein folding inhibitor 17-AAG, and the autophagy inhibitor chloroquine as exhibiting this property. AICAR induces p53-mediated apoptosis in primary mouse embryonic fibroblasts (MEFs) trisomic for chromosome 1, 13, 16, or 19. AICAR and 17-AAG, especially when combined, also show efficacy against aneuploid human cancer cell lines. Our results suggest that compounds that interfere with pathways that are essential for the survival of aneuploid cells could serve as a new treatment strategy against a broad spectrum of human tumors.
Assuntos
Aneuploidia , Antineoplásicos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Benzoquinonas/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Segregação de Cromossomos , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Lactamas Macrocíclicas/farmacologia , Camundongos , Neoplasias/tratamento farmacológico , Ribonucleotídeos/farmacologia , TrissomiaRESUMO
Photoreceptor loss is the final common endpoint in most retinopathies that lead to irreversible blindness, and there are no effective treatments to restore vision1,2. Chemical reprogramming of fibroblasts offers an opportunity to reverse vision loss; however, the generation of sensory neuronal subtypes such as photoreceptors remains a challenge. Here we report that the administration of a set of five small molecules can chemically induce the transformation of fibroblasts into rod photoreceptor-like cells. The transplantation of these chemically induced photoreceptor-like cells (CiPCs) into the subretinal space of rod degeneration mice (homozygous for rd1, also known as Pde6b) leads to partial restoration of the pupil reflex and visual function. We show that mitonuclear communication is a key determining factor for the reprogramming of fibroblasts into CiPCs. Specifically, treatment with these five compounds leads to the translocation of AXIN2 to the mitochondria, which results in the production of reactive oxygen species, the activation of NF-κB and the upregulation of Ascl1. We anticipate that CiPCs could have therapeutic potential for restoring vision.
Assuntos
Reprogramação Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Degeneração Retiniana/terapia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/transplante , Visão Ocular/efeitos dos fármacos , Animais , Proteína Axina/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Visão Ocular/fisiologiaRESUMO
mRNAs carry two layers of information, the genetic code and the information that dictates their post-transcriptional fate. The latter function relies on a complex interplay between cis-elements and trans-regulators, and unbiased identification of these elements is still challenging. To identify cis-elements that control gene expression, we use dimethyl sulfate (DMS) mutational profiling with sequencing and map changes in mRNA secondary structure following viral infection. Our dynamic structural data reveal a major role for ribosomes in unwinding secondary structures, which is further supported by the relationship we uncover between structure and translation efficiency. Moreover, our analysis revealed dozens of regions in viral and cellular mRNAs that exhibit changes in secondary structure. In-depth analysis of these regions reveals cis-elements in 3' UTRs that regulate mRNA stability and elements within coding sequences that control translation. Overall, our study demonstrates how mapping dynamic changes in mRNA structure allows unbiased identification of functional regulatory elements.
Assuntos
Citomegalovirus/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Fosfoproteínas/genética , Biossíntese de Proteínas , RNA Mensageiro/química , Proteínas da Matriz Viral/genética , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mutagênicos/farmacologia , Conformação de Ácido Nucleico , Fosfoproteínas/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais , Ésteres do Ácido Sulfúrico/farmacologia , Proteínas da Matriz Viral/metabolismoRESUMO
Proper control of the mitochondrial Ca2+ uniporter's pore (MCU) is required to allow Ca2+-dependent activation of oxidative metabolism and to avoid mitochondrial Ca2+ overload and cell death. The MCU's gatekeeping and cooperative activation is mediated by the Ca2+-sensing MICU1 protein, which has been proposed to form dimeric complexes anchored to the EMRE scaffold of MCU. We unexpectedly find that MICU1 suppresses inhibition of MCU by ruthenium red/Ru360, which bind to MCU's DIME motif, the selectivity filter. This led us to recognize in MICU1's sequence a putative DIME interacting domain (DID), which is required for both gatekeeping and cooperative activation of MCU and for cell survival. Thus, we propose that MICU1 has to interact with the D-ring formed by the DIME domains in MCU to control the uniporter.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Compostos de Rutênio/farmacologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genéticaRESUMO
The levels of copper, which is an essential element in living organisms, are under tight homeostatic control. Inactivating mutations in ATP7B, a P-type Cu-ATPase that functions in copper excretion, promote aberrant accumulation of the metal, primarily the in liver and brain. This condition underlies Wilson's disease, a severe autosomal recessive disorder characterized by profound hepatic and neurological deficits. Current treatment regimens rely on the use of broad specificity metal chelators as "decoppering" agents; however, there are side effects that limit their effectiveness. Here, we present the characterization of DPM-1001 {methyl 4-[7-hydroxy-10,13-dimethyl-3-({4-[(pyridin-2-ylmethyl)amino]butyl}amino)hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl] pentanoate} as a potent and highly selective chelator of copper that is orally bioavailable. Treatment of cell models, including fibroblasts derived from Wilson's disease patients, eliminated adverse effects associated with copper accumulation. Furthermore, treatment of the toxic milk mouse model of Wilson's disease with DPM-1001 lowered the levels of copper in the liver and brain, removing excess copper by excretion in the feces while ameliorating symptoms associated with the disease. These data suggest that it may be worthwhile to investigate DPM-1001 further as a new therapeutic agent for the treatment of Wilson's disease, with potential for application in other indications associated with elevated copper, including cancer and neurodegenerative diseases.
Assuntos
Quelantes/farmacologia , Cobre/metabolismo , Degeneração Hepatolenticular/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Linhagem Celular , Quelantes/uso terapêutico , Cobre/toxicidade , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Degeneração Hepatolenticular/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/patologia , CamundongosRESUMO
Chemical compounds have recently been introduced as alternative and non-integrating inducers of pluripotent stem cell fate. However, chemical reprogramming is hampered by low efficiency and the molecular mechanisms remain poorly characterized. Here, we show that inhibition of spleen tyrosine kinase (Syk) by R406 significantly promotes mouse chemical reprogramming. Mechanistically, R406 alleviates Syk / calcineurin (Cn) / nuclear factor of activated T cells (NFAT) signaling-mediated suppression of glycine, serine, and threonine metabolic genes and dependent metabolites. Syk inhibition upregulates glycine level and downstream transsulfuration cysteine biosynthesis, promoting cysteine metabolism and cellular hydrogen sulfide (H2 S) production. This metabolic rewiring decreased oxidative phosphorylation and ROS levels, enhancing chemical reprogramming. In sum, our study identifies Syk-Cn-NFAT signaling axis as a new barrier of chemical reprogramming and suggests metabolic rewiring and redox homeostasis as important opportunities for controlling cell fates.
Assuntos
Fibroblastos/metabolismo , Sulfeto de Hidrogênio/metabolismo , Quinase Syk/antagonistas & inibidores , Animais , Calcineurina/metabolismo , Células Cultivadas , Cisteína/metabolismo , Fibroblastos/efeitos dos fármacos , Glicina/metabolismo , Camundongos , Fatores de Transcrição NFATC/metabolismo , Oxazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine.
Assuntos
Diferenciação Celular , Fibroblastos/citologia , Cartilagem Hialina/citologia , Regeneração , Engenharia Tecidual/métodos , Animais , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrogênese , Fibroblastos/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento/farmacologia , Cartilagem Hialina/metabolismo , Cartilagem Hialina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
The innate immune system senses viral DNA that enters mammalian cells, or in aberrant situations self-DNA, and triggers type I interferon production. Here we present an integrative approach that combines quantitative proteomics, genomics and small molecule perturbations to identify genes involved in this pathway. We silenced 809 candidate genes, measured the response to dsDNA and connected resulting hits with the known signaling network. We identified ABCF1 as a critical protein that associates with dsDNA and the DNA-sensing components HMGB2 and IFI204. We also found that CDC37 regulates the stability of the signaling molecule TBK1 and that chemical inhibition of the CDC37-HSP90 interaction and several other pathway regulators potently modulates the innate immune response to DNA and retroviral infection.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , DNA Viral/imunologia , Células Dendríticas/imunologia , Fibroblastos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Chaperoninas/antagonistas & inibidores , Chaperoninas/genética , Chaperoninas/imunologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Citosol/virologia , DNA Viral/genética , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Regulação da Expressão Gênica/imunologia , Inativação Gênica , HIV-1/fisiologia , Proteína HMGB2/genética , Proteína HMGB2/imunologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/imunologia , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteômica , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Bibliotecas de Moléculas Pequenas/farmacologia , Vesiculovirus/fisiologiaRESUMO
Leber's hereditary optic neuropathy (LHON) is a visual impairment associated with mutations of mitochondrial genes encoding elements of the electron transport chain. While much is known about the genetics of LHON, the cellular pathophysiology leading to retinal ganglion cell degeneration and subsequent vision loss is poorly understood. The impacts of the G11778A mutation of LHON on bioenergetics, redox balance and cell proliferation were examined in patient-derived fibroblasts. Replacement of glucose with galactose in the culture media reveals a deficit in the proliferation of G11778A fibroblasts, imparts a reduction in ATP biosynthesis, and a reduction in capacity to accommodate exogenous oxidative stress. While steady-state ROS levels were unaffected by the LHON mutation, cell survival was diminished in response to exogenous H2O2.
Assuntos
DNA Mitocondrial , Fibroblastos , Galactose , Mutação , Atrofia Óptica Hereditária de Leber , Humanos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/metabolismo , Atrofia Óptica Hereditária de Leber/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Galactose/metabolismo , Mutação/genética , Proliferação de Células/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Células Cultivadas , Glucose/metabolismo , Glucose/farmacologiaRESUMO
Hypertrophic scar (HS) is a fibroproliferative skin disease characterized by abnormal wound healing and pathological excessive fibrosis of the skin. Currently, the molecular mechanism of the disease is still largely unknown, and there is no effective drug treatment. In this study, we explored the effect of Rynchopeterine on the formation of HS. HS fibroblasts (HSFs) were isolated from the HS tissues of patients recovering from severe burns. After treating HSFs with different concentrations of Rynchopeterine, CCK-8, EdU, and Annexin V-FITC/PI assays were used to detect the proliferation, apoptosis, and contractile ability of HSFs. RT-qPCR and Western blotting were performed to evaluate the effect of Rynchopeterine on the expression of miR-21 and hypoxia-inducible factor 1-alpha subunit suppressor (HIF1AN). The dual-luciferase reporter gene was used to verify the targeting relationship between miR-21 and HIF1AN. Rynchopeterine reduced the expression of Col1a2, Col3a1, and α-SMA, inhibited proliferation and contraction of HSFs, and increased apoptosis in a dose-dependent manner. miR-21 was highly expressed in HS tissues and HSFs, and Rynchopeterine could inhibit miR-21 expression. Overexpression of miR-21 and knockdown of HIF1AN increased proliferation, activation, contraction, and collagen synthesis of HSFs, and inhibited their apoptosis. In vivo, Rynchopeterine could reduce the collagen content of the dermis and the positive ratio of PCNA and α-SMA. Rynchopeterine is a good therapeutic agent for HS, which up-regulates the expression of HIF1AN by inhibiting miR-21, thereby inhibiting the formation of HS.