RESUMO
Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Neoplasias Hepáticas/metabolismo , Transdiferenciação Celular/genética , Carcinoma Hepatocelular/metabolismo , Mutação , Hepatócitos/metabolismo , Organoides/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genéticaRESUMO
BACKGROUND: Emerging studies have proved that colonic inflammation caused by refractory inflammatory bowel disease (IBD) can initiate the colitis-associated cancer (CAC), but the transition from inflammation to carcinoma is still largely unknown. METHODS: In this study, mouse colitis and CAC models were established, and the RNA-seq by circRNA microarray was employed to identify the differentially expressed circRNAs and mRNAs in different comparisons (DSS vs. NC and AOM/DSS vs. DSS). The bioinformatics analyses were used to search the common characteristics in mouse colitis and CAC. RESULTS: The K-means clustering algorithm packaged these differential expressed circRNAs into subgroup analysis, and the data strongly implied that mmu_circ_0001109 closely correlated to the pro-inflammatory signals, while mmu_circ_0001845 was significantly associated with the Wnt signalling pathway. Our subsequent data in vivo and in vitro confirmed that mmu_circ_0001109 could exacerbate the colitis by up-regulating the Jak-STAT3 and NF-kappa B signalling pathways, and mmu_circ_0001845 promoted the CAC transformation through the Wnt signalling pathway. By RNA blasting between mice and humans, the human RTEL1- and PRKAR2A-derived circRNAs, which might be considered as homeotic circRNAs of mmu_circ_0001109 and mmu_circ_0001845, respectively, were identified. The clinical data revealed that RTEL1-derived circRNAs had no clinical significance in human IBD and CAC. However, three PRKAR2A-derived circRNAs, which had the high RNA similarities to mmu_circ_0001845, were remarkably up-regulated in CAC tissue samples and promoted the transition from colitis to CAC. CONCLUSIONS: Our results suggested that these human PRKAR2A-derived circRNAs could be novel candidates for distinguishing CAC patients and predicted the prognosis of CAC.
Assuntos
Colite/complicações , Neoplasias Colorretais/classificação , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/efeitos adversos , Neoplasias/classificação , Animais , Colite/genética , Neoplasias Colorretais/etiologia , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Camundongos , Neoplasias/etiologia , RNA CircularRESUMO
Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Glioblastoma/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Glioblastoma/genética , Glioblastoma/fisiopatologia , Glioma/genética , Glioma/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução Genética/métodosRESUMO
Protein kinase A (PKA) plays an important role in regulating inflammation via its catalytic subunits. Recently, PKA regulatory subunits have been reported to directly modulate some signaling pathways and alleviate inflammation. However, the role of PKA regulatory subunits in colonic inflammation remains unclear. Therefore, we conducted this study to investigate the role of the PKA regulatory subunit PRKAR2A in colitis. We observed that PRKAR2A deficiency protected mice from dextran sulfate sodium (DSS)-induced experimental colitis. Our experiments revealed that the intestinal epithelial cell-specific deletion of Prkar2a contributed to this protection. Mechanistically, the loss of PRKAR2A in Prkar2a-/- mice resulted in an increased IFN-stimulated gene (ISG) expression and altered gut microbiota. Inhibition of ISGs partially reversed the protective effects against DSS-induced colitis in Prkar2a-/- mice. Antibiotic treatment and cross-fostering experiments demonstrated that the protection against DSS-induced colitis in Prkar2a-/- mice was largely dependent on the gut microflora. Altogether, our work demonstrates a previously unidentified function of PRKAR2A in promoting DSS-induced colitis.
Assuntos
Colite/etiologia , Colite/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/deficiência , Microbioma Gastrointestinal , Regulação da Expressão Gênica , Imunomodulação , Fatores Reguladores de Interferon/genética , Animais , Colite/patologia , Modelos Animais de Doenças , Microbioma Gastrointestinal/imunologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Fatores Reguladores de Interferon/metabolismo , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic ß-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human ß-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human ß-cells, it plays a major role at the translational level.
Assuntos
Metabolismo Energético/genética , Glucose/farmacologia , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas/genética , Linhagem Celular , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pró-Proteína Convertase 1/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
The habenula (Hb) is a bilateral, evolutionarily conserved epithalamic structure connecting forebrain and midbrain structures that has gained attention for its roles in depression, addiction, rewards processing, and motivation. Of its 2 major subdivisions, the medial Hb (MHb) and lateral Hb (LHb), MHb circuitry and function are poorly understood relative to those of the LHb. Prkar2a codes for cAMP-dependent protein kinase (PKA) regulatory subunit IIα (RIIα), a component of the PKA holoenzyme at the center of one of the major cell-signaling pathways conserved across systems and species. Type 2 regulatory subunits (RIIα, RIIß) determine the subcellular localization of PKA, and unlike other PKA subunits, Prkar2a has minimal brain expression except in the MHb. We previously showed that RIIα-knockout (RIIα-KO) mice resist diet-induced obesity. In the present study, we report that RIIα-KO mice have decreased consumption of palatable, "rewarding" foods and increased motivation for voluntary exercise. Prkar2a deficiency led to decreased habenular PKA enzymatic activity and impaired dendritic localization of PKA catalytic subunits in MHb neurons. Reexpression of Prkar2a in the Hb rescued this phenotype, confirming differential roles for Prkar2a in regulating the drives for palatable foods and voluntary exercise. Our findings show that in the MHb decreased PKA signaling and dendritic PKA activity decrease motivation for palatable foods, while enhancing the motivation for exercise, a desirable combination of behaviors.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Comportamento Alimentar/fisiologia , Habenula/metabolismo , Animais , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Habenula/fisiologia , Holoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Motivação/genética , Neurônios/metabolismo , Fenótipo , Condicionamento Físico Animal/fisiologiaRESUMO
Epithelioid fibrous histiocytoma (EFH) is a rare, benign, cutaneous neoplasm. This fibrohistiocytic tumor was once believed to be a variant of fibrous histiocytoma, but EFH is now known to be a distinct entity based on the presence of ALK gene rearrangements in most cases. The pattern of immunohistochemical expression of ALK in EFH in the literature thus far describes both granular cytoplasmic staining and nuclear staining. We present a case of EFH with dot-like Golgi pattern perinuclear ALK expression, a previously undescribed staining pattern. We surmised this unique staining pattern could be due to a novel fusion partner, and using FISH, we confirmed a rearrangement of the ALK (2p23) locus. Further investigation with whole transcriptome sequencing led to the discovery of PRKAR2A-ALK fusion, and the function of this fusion partner reflects a Golgi-predominant localization of the protein. Attention to the distinct immunohistochemical pattern of ALK expression may provide clues to the function of the fusion partner.
Assuntos
Quinase do Linfoma Anaplásico/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Histiocitoma Fibroso Benigno/genética , Neoplasias Cutâneas/genética , Adulto , Células Epitelioides/patologia , Feminino , Histiocitoma Fibroso Benigno/patologia , Humanos , Fusão Oncogênica , Proteínas de Fusão Oncogênica/genética , Neoplasias Cutâneas/patologiaRESUMO
Overactivation of the cAMP signal transduction pathway plays a central role in the pathogenesis of endocrine tumors. Genetic aberrations leading to increased intracellular cAMP or directly affecting PKA subunit expression have been identified in inherited and sporadic endocrine tumors, but are rare indicating the presence of nongenomic pathological PKA activation. In the present study, we examined the impact of hypoxia on PKA activation using human growth hormone (GH)-secreting pituitary tumors as a model of an endocrine disease displaying PKA-CREB overactivation. We show that hypoxia activates PKA and enhances CREB transcriptional activity and subsequently GH oversecretion. This is due to a previously uncharacterized ability of HIF-1α to suppress the transcription of the PKA regulatory subunit 2B (PRKAR2B) by sequestering Sp1 from the PRKAR2B promoter. The present study reveals a novel mechanism through which the transcription factor HIF-1α transduces environmental signals directly onto PKA activity, without affecting intracellular cAMP concentrations. By identifying a point of interaction between the cellular microenvironment and intracellular enzyme activation, neoplastic, and nonneoplastic diseases involving overactivated PKA pathway may be more efficiently targeted.
Assuntos
Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hipofisárias/genética , Ativação Transcricional/genética , Linhagem Celular Tumoral , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imunoglobulinas/genética , Fosforilação/genética , Neoplasias Hipofisárias/patologia , Transdução de Sinais/genética , Hipóxia Tumoral/genéticaRESUMO
BACKGROUND & AIMS: DNAJB1-PRKACA fusion is a specific driver event in fibrolamellar carcinoma (FLC), a rare subtype of hepatocellular carcinoma (HCC) that occurs in adolescents and young adults. In older patients, molecular determinants of HCC with mixed histological features of HCC and FLC (mixed-FLC/HCC) remain to be discovered. METHODS: A series of 151 liver tumors including 126 HCC, 15 FLC, and 10 mixed-FLC/HCC were analyzed by RNAseq and whole-genome- or whole-exome sequencing. Western blots were performed to validate genomic discoveries. Results were validated using the TCGA database. RESULTS: Most of the mixed-FLC/HCC RNAseq clustered in a robust subgroup of 17 tumors, which all had mutations or translocations inactivating BAP1, the gene encoding BRCA1-associated protein-1. Like FLC, BAP1-HCC were significantly enriched in females, patients with a lack of chronic liver disease, and fibrotic tumors compared to non-BAP1 HCC. However, patients were older and had a poorer prognosis than those with FLC. BAP1 tumors were immune hot, showed progenitor features and did not show DNAJB1-PRKACA fusion, while almost none of these tumors had mutations in CTNNB1, TP53 and TERT promoter. In contrast, 80% of the BAP1 tumors showed a chromosome gain of PRKACA at 19p13, combined with a loss of PRKAR2A (coding for the inhibitory regulatory subunit of PKA) at 3p21, leading to a high PRKACA/PRKAR2A ratio at the mRNA and protein levels. CONCLUSION: We have characterized a subgroup of BAP1-driven HCC with fibrolamellar-like features and a dysregulation of the PKA pathway, which could be at the root of the clinical and histological similarities between BAP1 tumors and DNAJB1-PRKACA FLCs. LAY SUMMARY: Herein, we have defined a homogeneous subgroup of hepatocellular carcinomas in which the BAP1 gene is inactivated. This leads to the development of cancers with features similar to those of fibrolamellar carcinoma. These tumors more frequently develop in females without chronic liver disease or cirrhosis. The presence of PKA activation and T cell infiltrates suggest that these tumors could be treated with PKA inhibitors or immunomodulators.
Assuntos
Carcinoma Hepatocelular/genética , Deleção Cromossômica , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Deleção de Genes , Neoplasias Hepáticas/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/patologia , Cromossomos Humanos Par 19/genética , Estudos de Coortes , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Transcriptoma , Adulto JovemRESUMO
Type II isoforms of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA-II) contain a phosphorylatable epitope within the inhibitory domain of RII subunits (pRII) with still unclear function. In vitro, RII phosphorylation occurs in the absence of cAMP, whereas staining of cells with pRII-specific antibodies revealed a cAMP-dependent pattern. In sensory neurons, we found that increased pRII immunoreactivity reflects increased accessibility of the already phosphorylated RII epitope during cAMP-induced opening of the tetrameric RII2:C2 holoenzyme. Accordingly, induction of pRII by cAMP was sensitive to novel inhibitors of dissociation, whereas blocking catalytic activity was ineffective. Also in vitro, cAMP increased the binding of pRII antibodies to RII2:C2 holoenzymes. Identification of an antibody specific for the glycine-rich loop of catalytic subunits facing the pRII-epitope confirmed activity-dependent binding with similar kinetics, proving that the reassociation is rapid and precisely controlled. Mechanistic modeling further supported that RII phosphorylation precedes cAMP binding and controls the inactivation by modulating the reassociation involving the coordinated action of phosphodiesterases and phosphatases.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Subunidades Proteicas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Anticorpos/farmacologia , Extratos Celulares , Permeabilidade da Membrana Celular/efeitos dos fármacos , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Isoenzimas/metabolismo , Masculino , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The main function of the A kinase-anchoring proteins (AKAPs) is to target the cyclic AMP-dependent protein kinase A (PKA) to its cellular substrates through the interaction with its regulatory subunits. Besides anchoring of PKA, AKAP8 participates in regulating the histone H3 lysine 4 (H3K4) histone methyltransferase (HMT) complexes. It is also involved in DNA replication, apoptosis, transcriptional silencing of rRNA genes, alternative splicing, and chromatin condensation during mitosis. In this study, we focused on the interaction between AKAP8 and the core subunit of all known H3K4 HMT complexes-DPY30 protein. Here, we demonstrate that the PKA-binding domain of AKAP8 and the C-terminal domain of DPY30, also called Dpy-30 motif, are crucial for the interaction between these proteins. We show that a single amino acid substitution in DPY30 L69D affects its dimerization and completely abolishes its interaction with AKAP8 and another DPY30-binding partner brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1), which is also AKAP domain-containing protein. We further demonstrate that AKAP8 interacts with DPY30 and the RII alpha regulatory subunit of PKA both in the interphase and in mitotic cells, and we show evidences that AKAP8L, a homologue of AKAP8, interacts with core subunits of the H3K4 HMT complexes, which suggests its role as a potential regulator of these complexes. The results presented here reinforce the analogy between AKAP8-RII alpha and AKAP8-DPY30 interactions, postulated before, and improve our understanding of the complexity of the cellular functions of the AKAP8 protein.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ancoragem à Quinase A/química , Ciclo Celular , Nucléolo Celular/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Dimerização , Genes Reporter , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Código das Histonas , Histona Metiltransferases/metabolismo , Humanos , Metilação , Modelos Moleculares , Proteínas Nucleares/química , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TranscriçãoRESUMO
t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway and activation of protein kinase A (PKA). The mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines overexpressing t-Darpp (SK.tDp) or a T39A phosphorylation mutant (SK.tDpT39A), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. SK.tDp cells had elevated PKA activity and showed diminished association of RI with PKAc, whereas SK.tDpT39A cells did not have these properties. Moreover, wild type t-Darpp associates with RI. Concurrent expression of Darpp-32 reversed t-Darrp's effects on PKA holoenzyme state, consistent with earlier observations that Darpp-32 reverses t-Darpp's activation of PKA. Together, t-Darpp phosphorylation at T39 seems to be crucial for t-Darpp-mediated PKA activation and this activation appears to occur through an association with RI and sequestering of RI away from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Neoplasias/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Dopamina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação , Receptor ErbB-2/genética , Trastuzumab/efeitos adversos , Trastuzumab/uso terapêuticoRESUMO
Pseudomonas aeruginosa is an opportunistic bacterium which induces some complications in immunocompromised patients. Pseudomonas aeruginosa is a quorum-sensing using bacterium which regulates its genes expression. The bacterium uses two famous pathways for quorum sensing entitled LasI/LasR and RhlI/RhlR systems. It has been documented that the bacteria which use quorum sensing are able to overcome immune responses. This review article aims to present recent information regarding the effects of Pseudomonas aeruginosa quorum sensing systems on the host immune responses.
Assuntos
Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Percepção de Quorum , Animais , Proteínas de Bactérias/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Evasão da Resposta Imune , Imunidade Inata , Transativadores/metabolismoRESUMO
Biochemical and structural studies demonstrate that S100A1 is involved in a Ca2+-dependent interaction with the type 2α and type 2ß regulatory subunits of protein kinase A (PKA) (RIIα and RIIß) to activate holo-PKA. The interaction was specific for S100A1 because other calcium-binding proteins (i.e., S100B and calmodulin) had no effect. Likewise, a role for S100A1 in PKA-dependent signaling was established because the PKA-dependent subcellular redistribution of HDAC4 was abolished in cells derived from S100A1 knockout mice. Thus, the Ca2+-dependent interaction between S100A1 and the type 2 regulatory subunits represents a novel mechanism that provides a link between Ca2+ and PKA signaling, which is important for the regulation of gene expression in skeletal muscle via HDAC4 cytosolic-nuclear trafficking.
Assuntos
Sinalização do Cálcio , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Histona Desacetilases/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas S100/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Ativação Enzimática , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/enzimologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas S100/genéticaRESUMO
BACKGROUND: Ageing skeletal muscle undergoes chronic denervation, and the neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with ageing. Previous studies in various species have shown that with ageing, type II fast-twitch skeletal muscle fibres show more atrophy and NMJ deterioration than type I slow-twitch fibres. However, how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle fibre-type specific denervation at the NMJ could be critical to identifying novel treatments for sarcopenia. Cardiac troponin T (cTnT), the heart muscle-specific isoform of TnT, is a key component of the mechanisms of muscle contraction. It is expressed in skeletal muscle during early development, after acute sciatic nerve denervation, in various neuromuscular diseases and possibly in ageing muscle. Yet the subcellular localization and function of cTnT in skeletal muscle is largely unknown. METHODS: Studies were carried out on isolated skeletal muscles from mice, vervet monkeys, and humans. Immunoblotting, immunoprecipitation, and mass spectrometry were used to analyse protein expression, real-time reverse transcription polymerase chain reaction was used to measure gene expression, immunofluorescence staining was performed for subcellular distribution assay of proteins, and electromyographic recording was used to analyse neurotransmission at the NMJ. RESULTS: Levels of cTnT expression in skeletal muscle increased with ageing in mice. In addition, cTnT was highly enriched at the NMJ region-but mainly in the fast-twitch, not the slow-twitch, muscle of old mice. We further found that the protein kinase A (PKA) RIα subunit was largely removed from, while PKA RIIα and RIIß are enriched at, the NMJ-again, preferentially in fast-twitch but not slow-twitch muscle in old mice. Knocking down cTnT in fast skeletal muscle of old mice: (i) increased PKA RIα and reduced PKA RIIα at the NMJ; (ii) decreased the levels of gene expression of muscle denervation markers; and (iii) enhanced neurotransmission efficiency at NMJ. CONCLUSIONS: Cardiac troponin T at the NMJ region contributes to NMJ functional decline with ageing mainly in the fast-twitch skeletal muscle through interfering with PKA signalling. This knowledge could inform useful targets for prevention and therapy of age-related decline in muscle function.
Assuntos
Envelhecimento , Denervação Muscular , Fibras Musculares de Contração Rápida/metabolismo , Troponina T/metabolismo , Potenciais de Ação , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Chlorocebus aethiops , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Contração Muscular , Junção Neuromuscular/fisiologia , Transmissão Sináptica , Troponina T/genéticaRESUMO
The kinetic participation of macrophages is critical for inflammatory resolution and recovery from myocardial infarction (MI), particularly with respect to the transition from the M1 to the M2 phenotype; however, the underlying mechanisms are poorly understood. In this study, we found that the deletion of prostaglandin (PG) D2 receptor subtype 1 (DP1) in macrophages retarded M2 polarization, antiinflammatory cytokine production, and resolution in different inflammatory models, including the MI model. DP1 deletion up-regulated proinflammatory genes expression via JAK2/STAT1 signaling in macrophages, whereas its activation facilitated binding of the separated PKA regulatory IIα subunit (PRKAR2A) to the transmembrane domain of IFN-γ receptor, suppressed JAK2-STAT1 axis-mediated M1 polarization, and promoted resolution. PRKAR2A deficiency attenuated DP1 activation-mediated M2 polarization and resolution of inflammation. Collectively, PGD2-DP1 axis-induced M2 polarization facilitates resolution of inflammation through the PRKAR2A-mediated suppression of JAK2/STAT1 signaling. These observations indicate that macrophage DP1 activation represents a promising strategy in the management of inflammation-associated diseases, including post-MI healing.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Prostaglandina D2/metabolismo , Subunidades Proteicas/metabolismo , Animais , Ceco/patologia , Polaridade Celular , Modelos Animais de Doenças , Feminino , Deleção de Genes , Hidantoínas , Janus Quinase 2/metabolismo , Ligadura , Macrófagos/citologia , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Isquemia Miocárdica/patologia , Peritonite/patologia , Ligação Proteica , Punções , Receptores Imunológicos , Receptores de Interferon , Receptores de Prostaglandina/deficiência , Receptores de Prostaglandina/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Cicatrização , ZimosanRESUMO
BACKGROUND: Protein kinase A (PKA) is a holoenzyme that consists of a dimer of regulatory subunits and two inactive catalytic subunits that bind to the regulatory subunit dimer. Four regulatory subunits (RIα, RIß, RIIα, RIIß) and four catalytic subunits (Cα, Cß, Cγ, Prkx) have been described in the human and mouse genomes. Previous studies showed that complete inactivation of the Prkar1a subunit (coding for RIα) in the germline leads to embryonic lethality, while Prkar1a-deficient mice are viable and develop schwannomas, thyroid, and bone neoplasms, and rarely lymphomas and sarcomas. Mice with inactivation of the Prkar2a and Prkar2b genes (coding for RIIα and RIIß, respectively) are also viable but have not been studied for their susceptibility to any tumors. METHODS: Cohorts of Prkar1a (+/-) , Prkar2a (+/-) , Prkar2a (-/-) , Prkar2b (+/-) and wild type (WT) mice have been observed between 5 and 25 months of age for the development of hematologic malignancies. Tissues were studied by immunohistochemistry; tumor-specific markers were also used as indicated. Cell sorting and protein studies were also performed. RESULTS: Both Prkar2a (-/-) and Prkar2a (+/-) mice frequently developed hematopoietic neoplasms dominated by histiocytic sarcomas (HS) with rare diffuse large B cell lymphomas (DLBCL). Southern blot analysis confirmed that the tumors diagnosed histologically as DLBCL were clonal B cell neoplasms. Mice with other genotypes did not develop a significant number of similar neoplasms. CONCLUSIONS: Prkar2a deficiency predisposes to hematopoietic malignancies in vivo. RIIα's likely association with HS and DLBCL was hitherto unrecognized and may lead to better understanding of these rare neoplasms.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/deficiência , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Neoplasias Hematológicas/genética , Animais , Transformação Celular Neoplásica/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/deficiência , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/deficiência , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Neoplasias Hematológicas/patologia , Imunofenotipagem , Camundongos , Camundongos Knockout , Fenótipo , Fatores de TempoRESUMO
Carney Complex (CNC), a human genetic syndrome predisposing to multiple neoplasias, is associated with bone lesions such as osteochondromyxomas (OMX). The most frequent cause for CNC is PRKAR1A deficiency; PRKAR1A codes for type-I regulatory subunit of protein kinase A (PKA). Prkar1a(+/-) mice developed OMX, fibrous dysplasia-like lesions (FDL) and other tumors. Tumor tissues in these animals had increased PKA activity due to an unregulated PKA catalytic subunit and increased PKA type II (PKA-II) activity mediated by the PRKAR2A and PRKAR2B subunits. To better understand the effect of altered PKA activity on bone, we studied Prkar2a and Prkar2b knock out (KO) and heterozygous mice; none of these mice developed bone lesions. When Prkar2a(+/-) and Prkar2b(+/-) mice were used to generate Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) animals, bone lesions formed that looked like those of the Prkar1a(+/-) mice. However, better overall bone organization and mineralization and fewer FDL lesions were found in both double heterozygote groups, indicating a partial restoration of the immature bone structure observed in Prkar1a(+/-) mice. Further investigation indicated increased osteogenesis and higher new bone formation rates in both Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) mice with some minor differences between them. The observations were confirmed with a variety of markers and studies. PKA activity measurements showed the expected PKA-II decrease in both double heterozygote groups. Thus, haploinsufficiency for either of PKA-II regulatory subunits improved bone phenotype of mice haploinsufficient for Prkar1a, in support of the hypothesis that the PRKAR2A and PRKAR2B regulatory subunits were in part responsible for the bone phenotype of Prkar1a(+/-) mice.
Assuntos
Osso e Ossos/patologia , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Haploinsuficiência , Animais , Antígenos de Diferenciação/biossíntese , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Osso e Ossos/metabolismo , Calcificação Fisiológica , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteogênese , Fenótipo , Isoformas de Proteínas/metabolismo , Células Tumorais CultivadasRESUMO
Our previous study reported that retinoic acid induced 16 (RAI16) could enhance tumorigenesis in hepatocellular carcinoma (HCC). However, the cellular functions of RAI16 are still unclear. In this study, by immunoprecipitation and tandem (MS/MS) mass spectrometry analysis, we identified that RAI16 interacted with the type II regulatory subunit of PKA (PKA-RIIα), acting as a novel protein kinase A anchoring protein (AKAP). In addition, RAI16 also interacted with heat shock protein 70 (HSP70) and 14-3-3θ. Further studies indicated that RAI16 mediated PKA phosphorylation of HSP70 at serine 486, resulting in anti-apoptosis events. RAI16 was also phosphorylated by the anchored PKA at serine 325, which promoted the recruitment of 14-3-3θ, which, in turn, inhibited RAI16 mediated PKA phosphorylation of HSP70. These findings offer mechanism insight into RAI16 mediated anti-apoptosis signaling in HCC.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas/metabolismo , Apoptose/fisiologia , Carcinoma Hepatocelular/patologia , Linhagem Celular , Sobrevivência Celular , Transformação Celular Neoplásica , Células HEK293 , Humanos , Neoplasias Hepáticas/patologia , Fosforilação , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
Protein kinase activity is regulated not only by direct strategies affecting activity but also by spatial and temporal regulatory mechanisms. Kinase signaling pathways are coordinated by scaffolding proteins that orchestrate the assembly of multi-protein complexes. One family of such scaffolding proteins are the A-kinase anchoring proteins (AKAPs). AKAPs share the commonality of binding cAMP-dependent protein kinase (PKA). In addition, they bind further signaling proteins and kinase substrates and tether such multi-protein complexes to subcellular locations. The A-kinase binding (AKB) domain of AKAPs typically contains a conserved helical motif that interacts directly with the dimerization/docking (D/D) domain of the regulatory subunits of PKA. Based on a pull-down proteomics approach, we identified neurochondrin (neurite-outgrowth promoting protein) as a previously unidentified AKAP. Here, we show that neurochondrin interacts directly with PKA through a novel mechanism that involves two distinct binding regions. In addition, we demonstrate that neurochondrin has strong isoform selectivity towards the RIIα subunit of PKA with nanomolar affinity. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.