RESUMO
N-myristoyltransferase (NMT) attaches the fatty acid myristate to the N-terminal glycine of proteins to sort them into soluble and membrane-bound fractions. Function of the energy-sensing AMP-activated protein kinase, AMPK, is myristoylation dependent. In rheumatoid arthritis (RA), pathogenic T cells shift glucose away from adenosine tri-phosphate production toward synthetic and proliferative programs, promoting proliferation, cytokine production, and tissue invasion. We found that RA T cells had a defect in NMT1 function, which prevented AMPK activation and enabled unopposed mTORC1 signaling. Lack of the myristate lipid tail disrupted the lysosomal translocation and activation of AMPK. Instead, myristoylation-incompetent RA T cells hyperactivated the mTORC1 pathway and differentiated into pro-inflammatory TH1 and TH17 helper T cells. In vivo, NMT1 loss caused robust synovial tissue inflammation, whereas forced NMT1 overexpression rescued AMPK activation and suppressed synovitis. Thus, NMT1 has tissue-protective functions by facilitating lysosomal recruitment of AMPK and dampening mTORC1 signaling.
Assuntos
Proteínas Quinases Ativadas por AMP/imunologia , Aciltransferases/imunologia , Artrite Reumatoide/imunologia , Sinovite/imunologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Adulto , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Células Cultivadas , Ativação Enzimática/imunologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Interferência de RNA , Sinovite/genética , Sinovite/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto JovemRESUMO
Pathogenic T cells in individuals with rheumatoid arthritis (RA) infiltrate non-lymphoid tissue sites, maneuver through extracellular matrix and form lasting inflammatory microstructures. Here we found that RA T cells abundantly express the podosome scaffolding protein TKS5, which enables them to form tissue-invasive membrane structures. TKS5 overexpression was regulated by the intracellular metabolic environment of RA T cells-specifically, by reduced glycolytic flux that led to deficiencies in ATP and pyruvate. ATPlopyruvatelo conditions triggered fatty acid biosynthesis and the formation of cytoplasmic lipid droplets. Restoration of pyruvate production or inhibition of fatty acid synthesis corrected the tissue-invasiveness of RA T cells in vivo and reversed their proarthritogenic behavior. Thus, metabolic control of T cell locomotion provides new opportunities to interfere with T cell invasion into specific tissue sites.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Artrite Psoriásica/metabolismo , Artrite Reumatoide/metabolismo , Linfócitos T/metabolismo , Trifosfato de Adenosina/metabolismo , Artrite Psoriásica/imunologia , Artrite Reumatoide/imunologia , Movimento Celular/imunologia , Ácidos Graxos/biossíntese , Feminino , Perfilação da Expressão Gênica , Glicólise/imunologia , Humanos , Immunoblotting , Imuno-Histoquímica , Inflamação , Masculino , Pessoa de Meia-Idade , Ácido Pirúvico/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Linfócitos T/imunologiaRESUMO
Single domain antibodies ("nanobodies") derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, as well as therapeutic and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research - i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat IgG. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.
RESUMO
Immune aging manifests with a combination of failing adaptive immunity and insufficiently restrained inflammation. In patients with rheumatoid arthritis (RA), T cell aging occurs prematurely, but the mechanisms involved and their contribution to tissue-destructive inflammation remain unclear. We found that RA CD4+ T cells showed signs of aging during their primary immune responses and differentiated into tissue-invasive, proinflammatory effector cells. RA T cells had low expression of the double-strand-break repair nuclease MRE11A, leading to telomeric damage, juxtacentromeric heterochromatin unraveling, and senescence marker upregulation. Inhibition of MRE11A activity in healthy T cells induced the aging phenotype, whereas MRE11A overexpression in RA T cells reversed it. In human-synovium chimeric mice, MRE11Alow T cells were tissue-invasive and pro-arthritogenic, and MRE11A reconstitution mitigated synovitis. Our findings link premature T cell aging and tissue-invasiveness to telomere deprotection and heterochromatin unpacking, identifying MRE11A as a therapeutic target to combat immune aging and suppress dysregulated tissue inflammation.
Assuntos
Artrite Reumatoide/imunologia , Senescência Celular/imunologia , Proteínas de Ligação a DNA/imunologia , Desoxirribonucleases/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Dano ao DNA/imunologia , Reparo do DNA/imunologia , Feminino , Humanos , Inflamação/imunologia , Leucócitos Mononucleares/imunologia , Camundongos , Sinovite/imunologia , Telômero/imunologia , Regulação para Cima/imunologiaRESUMO
Mitochondrial antiviral signaling platform protein (MAVS) acts as a central hub for RIG-I receptor proximal signal propagation. However, key components in the assembly of the MAVS mitochondrial platform that promote RIG-I mitochondrial localization and optimal activation are still largely undefined. Employing pooled RNAi and yeast two-hybrid screenings, we report that the mitochondrial adaptor protein tripartite motif (TRIM)14 provides a docking platform for the assembly of the mitochondrial signaling complex required for maximal activation of RIG-I-mediated signaling, consisting of WHIP and protein phosphatase PPP6C. Following viral infection, the ubiquitin-binding domain in WHIP bridges RIG-I with MAVS by binding to polyUb chains of RIG-I at lysine 164. The ATPase domain in WHIP contributes to stabilization of the RIG-I-dsRNA interaction. Moreover, phosphatase PPP6C is responsible for RIG-I dephosphorylation. Together, our findings define the WHIP-TRIM14-PPP6C mitochondrial signalosome required for RIG-I-mediated innate antiviral immunity.
Assuntos
Proteínas de Transporte/imunologia , Proteína DEAD-box 58/imunologia , Proteínas de Ligação a DNA/imunologia , Imunidade Inata , Mitocôndrias/imunologia , Proteínas Mitocondriais/imunologia , Complexos Multiproteicos/imunologia , Fosfoproteínas Fosfatases/imunologia , Transdução de Sinais/imunologia , ATPases Associadas a Diversas Atividades Celulares , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteína DEAD-box 58/genética , Proteínas de Ligação a DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Complexos Multiproteicos/genética , Fosfoproteínas Fosfatases/genética , Receptores Imunológicos , Transdução de Sinais/genética , Proteínas com Motivo Tripartido , Células Vero , Viroses/genética , Viroses/imunologia , Vírus/genética , Vírus/imunologiaRESUMO
Phospholipase A2 receptor 1 (PLA2R1) plays a crucial role in various diseases, including membranous nephropathy. However, the precise implications of PLA2R1 deficiency remain poorly understood. In this study, we created PLA2R1 knockout rats to explore potential consequences resulting from the loss of the PLA2R1 gene. Unexpectedly, our PLA2R1 knockout rats exhibited symptoms resembling those of chronic kidney disease after an 8-week observation period. Notably, several rats developed persistent proteinuria, a hallmark of renal dysfunction. Immunohistochemical and immunofluorescence analyses revealed insignificant glomerular fibrosis, reduced podocyte count, and augmented glomerular expression of complement C3 (C3) compared to immunoglobin A (IgA) and immunoglobin G(IgG) in the rat model. These findings suggest that the loss of PLA2R1 may contribute to the pathogenesis of membranous nephropathy and related conditions. Our knockout rat model provides a valuable tool for investigating the underlying pathology of PLA2R1-associated diseases, and may facilitate the development of targeted therapies for membranous nephropathy and other related disorders.
Assuntos
Glomerulonefrite Membranosa , Receptores da Fosfolipase A2 , Animais , Ratos , Autoanticorpos , Glomerulonefrite Membranosa/genética , Glomerulonefrite Membranosa/diagnóstico , Glomerulonefrite Membranosa/metabolismo , Receptores da Fosfolipase A2/genética , Receptores da Fosfolipase A2/metabolismoRESUMO
Stringent control of the type I interferon signaling pathway is important for maintaining host immune responses and homeostasis, yet the molecular mechanisms responsible for its tight regulation are still poorly understood. Here we report that the pattern-recognition receptor NLRP4 regulated the activation of type I interferon mediated by double-stranded RNA or DNA by targeting the kinase TBK1 for degradation. NLRP4 recruited the E3 ubiquitin ligase DTX4 to TBK1 for Lys48 (K48)-linked polyubiquitination at Lys670, which led to degradation of TBK1. Knockdown of either DTX4 or NLRP4 abrogated K48-linked ubiquitination and degradation of TBK1 and enhanced the phosphorylation of TBK1 and the transcription factor IRF3. Our results identify a previously unrecognized role for NLRP4 in the regulation of type I interferon signaling and provide molecular insight into the mechanisms by which NLRP4-DTX4 targets TBK1 for degradation.
Assuntos
Interferon Tipo I/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata/imunologia , Immunoblotting , Imunoprecipitação , Interferon Tipo I/imunologia , Fosforilação , Proteínas Serina-Treonina Quinases/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/imunologia , Transfecção , Ubiquitina-Proteína Ligases/imunologia , UbiquitinaçãoRESUMO
A facile and efficient copper-catalyzed domino-double annulation strategy was developed from easily accessible o-aminobenzamides and 2-iodoisothiocyanates, which affords a direct pathway for the synthesis of tetracyclic fused 12H-benzo[4,5]thiazolo[2,3-b]quinazolin-12-ones in moderate to good yields without the addition of ligands, bases, and external oxidants. The reaction involves a C-N bond cleavage and the formation of a C-N/C-S bond in one step with the advantages of using an inexpensive copper catalyst and easy operation. Mechanistic studies suggest that this transformation proceeds via intermolecular condensation of o-aminobenzamides with 2-iodoisothiocyanates, followed by an intramolecular Ullmann-type cross-coupling cyclization reaction.
RESUMO
A palladium-catalyzed intermolecular [2 + 2 + 2] oxidative coupling-annulation of terminal alkenes and alkynes using copper(II) as the oxidant has been developed through direct C-C bond formation. These reactions provide effective access to multiaryl-substituted benzenes with high regioselectivity in the absence of any ligands. The features of this protocol are broad substrate scope, and high atom and step economy. The aggregation-induced emission properties of selected products were further investigated. These synthesized multiaryl-substituted benzenes may be worth exploring for further applications in the fields of advanced functional materials or drugs.
RESUMO
DNA double-strand breaks repaired by non-homologous end joining display limited DNA end-processing and chromosomal mobility. By contrast, double-strand breaks undergoing homology-directed repair exhibit extensive processing and enhanced motion. The molecular basis of this movement is unknown. Here, using Xenopus laevis cell-free extracts and mammalian cells, we establish that nuclear actin, WASP, and the actin-nucleating ARP2/3 complex are recruited to damaged chromatin undergoing homology-directed repair. We demonstrate that nuclear actin polymerization is required for the migration of a subset of double-strand breaks into discrete sub-nuclear clusters. Actin-driven movements specifically affect double-strand breaks repaired by homology-directed repair in G2 cell cycle phase; inhibition of actin nucleation impairs DNA end-processing and homology-directed repair. By contrast, ARP2/3 is not enriched at double-strand breaks repaired by non-homologous end joining and does not regulate non-homologous end joining. Our findings establish that nuclear actin-based mobility shapes chromatin organization by generating repair domains that are essential for homology-directed repair in eukaryotic cells.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo de DNA por Recombinação , Xenopus laevis/genética , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Actinas/metabolismo , Animais , Extratos Celulares , Cromatina/metabolismo , Reparo do DNA por Junção de Extremidades , Feminino , Movimento , Ligação Proteica , Transporte Proteico , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Infertility is a global health problem affecting millions of people of reproductive age worldwide, with approximately half caused by males. Chitosan oligosaccharide (COS) has strong antioxidant capacity, but its impact on the male reproductive system has not been effectively evaluated. To address this, we integrated RNA-seq, serum metabolomics and intestinal 16â¯S rDNA analysis to conduct a comprehensive investigation on the male reproductive system. The results showed that COS has potential targets for the treatment of oligospermia, which can promote the expression of meiotic proteins DDX4, DAZL and SYCP1, benefit germ cell proliferation and testicular development, enhance antioxidant capacity, and increase the expression of testicular steroid proteins STAR and CYP11A1. At the same time, COS can activate PI3K-Akt signaling pathway in testis and TM3 cells. Microbiome and metabolomics analysis suggested that COS alters gut microbial community composition and cooperates with serum metabolites to regulate spermatogenesis. Therefore, COS promotes male reproduction by regulating intestinal microorganisms and serum metabolism, activating PI3K-Akt signaling pathway, improving testicular antioxidant capacity and steroid regulation.
RESUMO
Mosquito viruses cause unpredictable outbreaks of disease. Recently, several unassigned viruses isolated from mosquitoes, including the Omono River virus (OmRV), were identified as totivirus-like viruses, with features similar to those of the Totiviridae family. Most reported members of this family infect fungi or protozoans and lack an extracellular life cycle stage. Here, we identified a new strain of OmRV and determined high-resolution structures for this virus using single-particle cryo-electron microscopy. The structures feature an unexpected protrusion at the five-fold vertex of the capsid. Disassociation of the protrusion could result in several conformational changes in the major capsid. All these structures, together with some biological results, suggest the protrusions' associations with the extracellular transmission of OmRV.
Assuntos
Microscopia Crioeletrônica/métodos , Vírus de RNA de Cadeia Dupla/ultraestrutura , Proteínas Estruturais Virais/ultraestrutura , Aedes/virologia , AnimaisRESUMO
The enzymes are biological macromolecules that biocatalyze certain biochemical reactions without undergoing any modification or degradation at the end of the reaction. In this work, we constructed a recombinant novel Raoultella sp. NX-TZ-3-15 strain that produces heparinase with a maltose binding tag to enhance its production and activity. Additionally, MBP-heparinase was purified and its enzymatic capabilities are investigated to determine its industrial application. Moreover, the recombinant plasmid encoding the MBP-heparinase fusion protein was effectively generated and purified to a high purity. According to SDS-PAGE analysis, the MBP-heparinase has a molecular weight of around 70 kDa and the majority of it being soluble with a maximum activity of 5386 U/L. It has also been noted that the three ions of Ca2 + , Co2 + , and Mg2 + can have an effect on heparinase activities, with Mg2 + being the most noticeable, increasing by about 85%, while Cu2 + , Fe2 + , Zn2 + having an inhibitory effect on heparinase activities. Further investigations on the mechanistic action, structural features, and genomes of Raoultella sp. NX-TZ-3-15 heparinase synthesis are required for industrial-scale manufacturing.
Assuntos
Escherichia coli , Polissacarídeo-Liases , Enterobacteriaceae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Heparina Liase/química , Heparina Liase/genética , Heparina Liase/metabolismo , Plasmídeos/genética , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismoRESUMO
Intrahepatic cholangiocarcinoma (ICC), a type of bile duct cancer, has a high mortality rate. Gut microbiota, bile acid (BA) metabolism, and cytokines have not been characterized in patients with ICC, and better noninvasive diagnostic approaches for ICC are essential to be established. Therefore, in this study we aimed to improve our understanding of changes in gut microbiota, BA metabolism, and cytokines in patients with ICC. We found that the α-diversities and ß-diversities of ICC were highest and that the abundances of four genera (Lactobacillus, Actinomyces, Peptostreptococcaceae, and Alloscardovia) were increased in patients with ICC compared with those in patients with hepatocellular carcinoma or liver cirrhosis and in healthy individuals. The glycoursodeoxycholic acid and tauroursodeoxycholic acid (TUDCA) plasma-stool ratios were obviously increased in patients with ICC. Furthermore, the genera Lactobacillus and Alloscardovia that were positively correlated with TUDCA plasma-stool ratios were combined to discriminate ICC from the other three diseases. Vascular invasion (VI) frequently led to a poor prognosis in patients with ICC. Compared with patients with ICC without VI, patients with VI had a greater abundance of the family Ruminococcaceae, increased levels of plasma interleukin (IL)-4 and six conjugated BAs, and decreased levels of plasma IL-6 and chenodeoxycholic acid. A positive correlation between plasma taurocholic acid and IL-4 was observed in patients with ICC. Plasma TUDCA was negatively correlated with the abundance of the genus Pseudoramibacter and the survival time of patients with ICC, but had no effect on tumor size, as determined in two murine tumor models. Conclusion: In this study, we identified some biomarkers, including gut microbiota, BAs and inflammatory cytokines, for the diagnosis of ICC and prediction of VI in patients with ICC.
Assuntos
Ácidos e Sais Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Citocinas/sangue , Microbioma Gastrointestinal/fisiologia , Actinobacteria/isolamento & purificação , Animais , Neoplasias dos Ductos Biliares/imunologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/microbiologia , Colangiocarcinoma/imunologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/microbiologia , Humanos , Lactobacillus/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Invasividade NeoplásicaRESUMO
DNA double-strand breaks (DSBs) activate a DNA damage response (DDR) that coordinates checkpoint pathways with DNA repair. ATM and ATR kinases are activated sequentially. Homology-directed repair (HDR) is initiated by resection of DSBs to generate 3' single-stranded DNA overhangs. How resection and HDR are activated during DDR is not known, nor are the roles of ATM and ATR in HDR. Here, we show that CtIP undergoes ATR-dependent hyperphosphorylation in response to DSBs. ATR phosphorylates an invariant threonine, T818 of Xenopus CtIP (T859 in human). Nonphosphorylatable CtIP (T818A) does not bind to chromatin or initiate resection. Our data support a model in which ATM activity is required for an early step in resection, leading to ATR activation, CtIP-T818 phosphorylation, and accumulation of CtIP on chromatin. Chromatin binding by modified CtIP precedes extensive resection and full checkpoint activation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/fisiologia , Extratos Celulares/isolamento & purificação , Cromatina/metabolismo , Sequência Conservada , Clivagem do DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Coelhos , Proteínas Supressoras de Tumor/química , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/química , Proteínas de Xenopus/fisiologia , Xenopus laevisRESUMO
Melanoma differentiation-associated gene-5 (MDA5) recognizes distinct subsets of viruses including Encephalomyocarditis virus (EMCV) of picornavirus family, but the molecular mechanisms underlying the specificity of the viral recognition of MDA5 in immune cells remain obscure. DHX29 is an RNA helicase required for the translation of 5' structured mRNA of host and many picornaviruses (such as EMCV). We identify that DXH29 as a key RNA co-sensor, plays a significant role for specific recognition and triggering anti-EMCV immunity. We have observed that DHX29 regulates MDA5-, but not RIG-I-, mediated type I interferon signaling by preferentially interacting with structured RNAs and specifically with MDA5 for enhancing MDA5-dsRNA binding affinity. Overall, our results identify a critical role for DHX29 in innate immune response and provide molecular insights into the mechanisms by which DHX29 recognizes 5' structured EMCV RNA and interacts with MDA5 for potent type I interferon signaling and antiviral immunity.
Assuntos
Infecções por Cardiovirus/imunologia , Vírus da Encefalomiocardite/imunologia , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon/fisiologia , RNA Helicases/fisiologia , RNA Viral/imunologia , Animais , Infecções por Cardiovirus/genética , Células Cultivadas , Chlorocebus aethiops , Vírus da Encefalomiocardite/genética , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , RNA Helicases/genética , RNA Viral/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células VeroRESUMO
OBJECTIVE: To investigate the effect of paraquat (PQ) exposure on gene expression in neural stem cells as well as structures and functions of vascular endothelial cells. METHODS: RNA-Seq was used to explore the differentially expressed genes in human umbilical cord blood-neural stem cells (HUCB-NSCs) at different stages (eg, proliferation, early and late differentiation) in the presence of PQ. The effects of PQ on human umbilical vein endothelial cells (HUVECs), including cell proliferation, apoptosis, cytokines secretion, and expression of tight junction proteins, were assessed with CCK-8, flow cytometry, ELISA, and western blot analysis, individually. RESULTS: A total of 53 genes were up-regulated and 61 genes were down-regulated in PQ treated HUCB-NSCs, including seven genes associated with the differentiation of neural stem cells, for example, Gfap, S100B, Oct4, Gdf3, Sox1, Pax6, and Ngn1. PQ treatment significantly reduced the proliferation of HUVECs, inhibited cytokines secretion (VEGF, BFGF) and expressions of tight junction-associated protein (Claudin 1, Occludin, ZO-1), as well as induced significant apoptosis. CONCLUSION: Our study suggests that PQ impairs the development of nervous system by regulating the expression of genes associated with neural stem cell differentiation, as well as the structure and function of vascular endothelial cells, which together lead to abnormality in the nervous system.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Paraquat/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/genética , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo , Sequenciamento de Nucleotídeos em Larga Escala , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Células-Tronco Neurais/patologia , Proteínas de Junções Íntimas/genética , Regulação para CimaRESUMO
It remains particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here we present a strategy for isolating complexes at endogenous levels from GFP-tagged transgenic cell lines. Using cross-linking mass spectrometry, we extracted distance restraints that allowed us to model the complexes' molecular architectures.
Assuntos
Espectrometria de Massas/métodos , Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , Animais , Reagentes de Ligações Cruzadas/química , Eletroforese em Gel de Poliacrilamida , Exossomos/química , Proteínas de Fluorescência Verde/genética , Fígado/química , Camundongos Transgênicos , Complexos Multiproteicos/genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: Accelerated atherosclerotic disease typically complicates rheumatoid arthritis (RA), leading to premature cardiovascular death. Inflammatory macrophages are key effector cells in both rheumatoid synovitis and the plaques of coronary artery disease (CAD). Whether both diseases share macrophage-dependent pathogenic mechanisms is unknown. METHODS: Patients with RA or CAD (at least one myocardial infarction) and healthy age-matched controls were recruited into the study. Peripheral blood CD14+ monocytes were differentiated into macrophages. Metabolic profiles were assessed by Seahorse Analyzer, intracellular ATP concentrations were quantified and mitochondrial protein localisation was determined by confocal image analysis. RESULTS: In macrophages from patients with RA or CAD, mitochondria consumed more oxygen, generated more ATP and built tight interorganelle connections with the endoplasmic reticulum, forming mitochondria-associated membranes (MAM). Calcium transfer through MAM sites sustained mitochondrial hyperactivity and was dependent on inactivation of glycogen synthase kinase 3b (GSK3b), a serine/threonine kinase functioning as a metabolic switch. In patient-derived macrophages, inactivated pGSK3b-Ser9 co-precipitated with the mitochondrial fraction. Immunostaining of atherosclerotic plaques and synovial lesions confirmed that most macrophages had inactivated GSK3b. MAM formation and GSK3b inactivation sustained production of the collagenase cathepsin K, a macrophage effector function closely correlated with clinical disease activity in RA and CAD. CONCLUSIONS: Re-organisation of the macrophage metabolism in patients with RA and CAD drives unopposed oxygen consumption and ultimately, excessive production of tissue-destructive enzymes. The underlying molecular defect relates to the deactivation of GSK3b, which controls mitochondrial fuel influx and as such represents a potential therapeutic target for anti-inflammatory therapy.
Assuntos
Artrite Reumatoide/patologia , Doença da Artéria Coronariana/patologia , Quinases da Glicogênio Sintase/metabolismo , Macrófagos/metabolismo , Sinovite/patologia , Pesquisa Translacional Biomédica , Artrite Reumatoide/sangue , Artrite Reumatoide/fisiopatologia , Estudos de Casos e Controles , Células Cultivadas , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/fisiopatologia , Feminino , Humanos , Macrófagos/enzimologia , Masculino , Pessoa de Meia-Idade , Tamanho Mitocondrial/fisiologia , Monócitos/metabolismo , Consumo de Oxigênio/fisiologia , Fatores de Risco , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Sinovite/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a common digestive malignancy. MiR-223, a well-identified miRNA, exhibits diverse properties in different cancers. In this study, we demonstrated that miR-223 could suppress cell growth and promote apoptosis in HepG2 and Bel-7402 HCC cell lines. We screened and identified a novel miR-223 target, Ras-related protein Rab-1(Rab1). Upregulation of miR-223 would specifically and markedly down-regulate Rab1 expression. In addition, miR-223-overexpressing subclones showed significant cell growth inhibition by increasing cell apoptosis in HepG2 and Bel-7402 cells. To identify the mechanisms, we firstly investigated the mTOR pathway and found that pmTOR, p70S6K and Bcl-2 were dramatically down-regulated after miR-223 transfection, while no changes in the level of Bax was visualized. Furthermore, our data showed that the anti-tumor effects arising from miR-223 transfection in HCC cells may be due to the deactivation of mTOR pathway caused by the suppression of Rab1 expression when miR-223 is overexpressed. In summary, our results indicate that miR-223 functions as a tumor suppressor and plays a critical role in inhibiting the tumorigenesis and promoting the apoptosis of HCC through the mTOR signaling pathway in vitro. By targeting Rab1, miR-223 efficiently mediates the mTOR pathway. Given these, miR-223 may be a potential therapeutic target for treating HCC.