RESUMEN
Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1-10 µm), whereas higher levels (0.5-1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 µm to 1 mm H2O2), with maximal effects at 500 µm H2O2 H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 µm H2O2 However, higher H2O2 levels (≥50 µm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation.
Asunto(s)
Apoptosis , Ciclofilinas/fisiología , Mitocondrias/fisiología , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Necrosis , Estrés Oxidativo , Páncreas/patología , Células Acinares/metabolismo , Células Acinares/patología , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Peptidil-Prolil Isomerasa F , Metabolismo Energético , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL , Ratones Noqueados , Poro de Transición de la Permeabilidad Mitocondrial , Páncreas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: Caffeine reduces toxic Ca2+ signals in pancreatic acinar cells via inhibition of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated signalling, but effects of other xanthines have not been evaluated, nor effects of xanthines on experimental acute pancreatitis (AP). We have determined effects of caffeine and its xanthine metabolites on pancreatic acinar IP3R-mediated Ca2+ signalling and experimental AP. DESIGN: Isolated pancreatic acinar cells were exposed to secretagogues, uncaged IP3 or toxins that induce AP and effects of xanthines, non-xanthine phosphodiesterase (PDE) inhibitors and cyclic adenosine monophosphate and cyclic guanosine monophosphate (cAMP/cGMP) determined. The intracellular cytosolic calcium concentration ([Ca2+]C), mitochondrial depolarisation and necrosis were assessed by confocal microscopy. Effects of xanthines were evaluated in caerulein-induced AP (CER-AP), taurolithocholic acid 3-sulfate-induced AP (TLCS-AP) or palmitoleic acid plus ethanol-induced AP (fatty acid ethyl ester AP (FAEE-AP)). Serum xanthines were measured by liquid chromatography-mass spectrometry. RESULTS: Caffeine, dimethylxanthines and non-xanthine PDE inhibitors blocked IP3-mediated Ca2+ oscillations, while monomethylxanthines had little effect. Caffeine and dimethylxanthines inhibited uncaged IP3-induced Ca2+ rises, toxin-induced Ca2+ release, mitochondrial depolarisation and necrotic cell death pathway activation; cAMP/cGMP did not inhibit toxin-induced Ca2+ rises. Caffeine significantly ameliorated CER-AP with most effect at 25â mg/kg (seven injections hourly); paraxanthine or theophylline did not. Caffeine at 25â mg/kg significantly ameliorated TLCS-AP and FAEE-AP. Mean total serum levels of dimethylxanthines and trimethylxanthines peaked at >2â mM with 25â mg/kg caffeine but at <100â µM with 25â mg/kg paraxanthine or theophylline. CONCLUSIONS: Caffeine and its dimethylxanthine metabolites reduced pathological IP3R-mediated pancreatic acinar Ca2+ signals but only caffeine ameliorated experimental AP. Caffeine is a suitable starting point for medicinal chemistry.
Asunto(s)
Células Acinares/efectos de los fármacos , Cafeína/farmacología , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Páncreas/patología , Pancreatitis/prevención & control , Inhibidores de Fosfodiesterasa/farmacología , Células Acinares/metabolismo , Animales , Cafeína/uso terapéutico , Muerte Celular/efectos de los fármacos , Células Cultivadas , Ceruletida , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Citosol/metabolismo , Etanol , Ácidos Grasos Monoinsaturados , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Ratones , Microscopía Confocal , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Necrosis/diagnóstico por imagen , Pancreatitis/sangre , Pancreatitis/inducido químicamente , Inhibidores de Fosfodiesterasa/uso terapéutico , Transducción de Señal/efectos de los fármacos , Ácido Taurolitocólico/análogos & derivados , Xantinas/sangre , Xantinas/farmacologíaRESUMEN
OBJECTIVES: To evaluate the therapeutic potential of I-BET-762, an inhibitor of the bromodomain and extra-terminal (BET) protein family, in experimental acute pancreatitis (AP). METHODS: AP was induced by retrograde infusion of taurolithocholic acid sulphate into the biliopancreatic duct (TLCS-AP) or 2 intraperitoneal (i.p.) injections of ethanol and palmitoleic acid 1 h apart (FAEE-AP) or 12 hourly i.p. injections of caerulein (CER-AP). In all treatment groups, I-BET-762 (30 mg/kg, i.p.) was administered at the time of disease induction and again 12 h later. AP severity was assessed at 24 h by serum biochemistry, multiple cytokines and histopathology. RESULTS: TLCS-AP, FAEE-AP and CER-AP resulted in characteristic elevations in serum amylase and cytokine levels, increased pancreatic trypsin and myeloperoxidase activity, typical pancreatic histopathological changes and lung injury. Treatment with I-BET-762 significantly reduced biochemical, cytokine and histopathological responses in TLCS-AP and FAEE-AP, but not CER-AP. CONCLUSIONS: These results suggest that in different forms of AP there are significant differences in the epigenetic control of gene transcription contributing to the severity of disease responses. There is therapeutic potential in targeting bromodomains for the treatment of gallstone- and alcohol-related pancreatitis.
Asunto(s)
Benzodiazepinas/farmacología , Ácidos y Sales Biliares/toxicidad , Ceruletida/toxicidad , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Pancreatitis/inducido químicamente , Receptores de Superficie Celular/antagonistas & inhibidores , Ácido Taurolitocólico/análogos & derivados , Enfermedad Aguda , Amilasas/sangre , Amilasas/metabolismo , Animales , Citocinas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inflamación/prevención & control , Pulmón/enzimología , Masculino , Ratones , Páncreas/enzimología , Páncreas/patología , Pancreatitis/terapia , Peroxidasa/genética , Peroxidasa/metabolismo , Ácido Taurolitocólico/toxicidad , Tripsina/metabolismoRESUMEN
BACKGROUND & AIMS: Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. METHODS: Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. RESULTS: GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. CONCLUSIONS: Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.
Asunto(s)
Células Acinares/efectos de los fármacos , Benzamidas/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Calcio/metabolismo , Pancreatitis/tratamiento farmacológico , Pirazoles/farmacología , Células Acinares/citología , Enfermedad Aguda , Animales , Ácidos y Sales Biliares/toxicidad , Calcio/toxicidad , Células Cultivadas , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Indoles/toxicidad , Ratones , Ratones Endogámicos C57BL , Proteína ORAI1 , Pancreatitis/inducido químicamente , Pancreatitis/metabolismo , Tapsigargina/toxicidad , Factores de Tiempo , Resultado del TratamientoRESUMEN
Acute pancreatitis (AP) is a formidable disease, which, in severe forms, causes significant mortality. Biliary AP, or gallstone obstruction-associated AP, accounts for 30-50% of all clinical cases of AP. In biliary AP, pancreatic acinar cell (PAC) death (the initiating event in the disease) is believed to occur as acinar cells make contact with bile salts when bile refluxes into the pancreatic duct. Recent advances have unveiled an important receptor responsible for the major function of bile acids on acinar cells, namely, the cell surface G-protein-coupled bile acid receptor-1 (Gpbar1), located in the apical pole of the PAC. High concentrations of bile acids induce cytosolic Ca(2+) overload and inhibit mitochondrial adenosine triphosphate (ATP) production, resulting in cell injury to both PACs and pancreatic ductal epithelial cells. Various bile salts are employed to induce experimental AP, most commonly sodium taurocholate. Recent characterization of taurolithocholic acid 3-sulphate on PACs has led researchers to focus on this bile salt because of its potency in causing acinar cell injury at relatively low, sub-detergent concentrations, which strongly implicates action via the receptor Gpbar1. Improved surgical techniques have enabled the infusion of bile salts into the pancreatic duct to induce experimental biliary AP in mice, which allows the use of these transgenic animals as powerful tools. This review summarizes recent findings using transgenic mice in experimental biliary AP.
Asunto(s)
Colestasis/etiología , Cálculos Biliares/complicaciones , Conductos Pancreáticos/metabolismo , Pancreatitis/etiología , Enfermedad Aguda , Animales , Ácidos y Sales Biliares/metabolismo , Colestasis/genética , Colestasis/metabolismo , Colestasis/patología , Modelos Animales de Enfermedad , Cálculos Biliares/inducido químicamente , Cálculos Biliares/genética , Cálculos Biliares/metabolismo , Cálculos Biliares/patología , Genotipo , Humanos , Ratones , Ratones Transgénicos , Conductos Pancreáticos/patología , Pancreatitis/genética , Pancreatitis/metabolismo , Pancreatitis/patología , Fenotipo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Introduction: Multiple drug-resistant Gram-negative bacterial (MDR-GNB) bacteraemia poses a serious threat to patients in hospital. Infected pancreatic necrosis (IPN) patients are a vulnerable population to infectious complications during hospitalization. This study aims to evaluate the impact of MDR Gram-negative bacteraemia on IPN patients. Methods: A case-control study was performed with data collected from 1 January 2016 to 1 July 2022 in a Chinese tertiary teaching hospital. Clinical data of the IPN patients with MDR-GNB bacteraemia were analyzed and compared to those of a matched control group without MDR-GNB bacteraemia (case-control ratio of 1:2). Comparisons were performed between with/without MDR-GNB bacteraemia and different severities of acute pancreatitis (AP). Independent predictors of overall mortality were identified via univariate and multivariate binary logistic regression analyses. Results: MDR-GNB bacteraemia was related to a higher mortality rate (62.5% vs. 8.3%, p < 0.001). Severe AP combined with MDR-GNB bacteraemia further increased mortality up to 81.3% (p = 0.025). MDR-GNB bacteraemia (odds ratio (OR) = 8.976, 95% confidence interval (CI) = 1.805 -44.620, p = 0.007) and severe AP (OR = 9.414, 95% CI = 1.742 -50.873, p = 0.009) were independent predictors of overall mortality. MDR- Klebsiella pneumoniae was the most common causative pathogen. Conclusion: A higher mortality rate in IPN patients was related to MDR-GNB bacteraemia and further increased in severe AP patients combined with MDR-GNB bacteraemia.
Asunto(s)
Bacteriemia , Pancreatitis Aguda Necrotizante , Humanos , Pancreatitis Aguda Necrotizante/complicaciones , Enfermedad Aguda , Estudios de Casos y Controles , Hospitales de EnseñanzaRESUMEN
BACKGROUND: Acute pancreatitis (AP) is a potentially severe or even fatal inflammation of the pancreas. Early identification of patients at high risk for developing a severe course of the disease is crucial for preventing organ failure and death. Most of the former predictive scores require many parameters or at least 24 h to predict the severity; therefore, the early therapeutic window is often missed. METHODS: The early achievable severity index (EASY) is a multicentre, multinational, prospective and observational study (ISRCTN10525246). The predictions were made using machine learning models. We used the scikit-learn, xgboost and catboost Python packages for modelling. We evaluated our models using fourfold cross-validation, and the receiver operating characteristic (ROC) curve, the area under the ROC curve (AUC), and accuracy metrics were calculated on the union of the test sets of the cross-validation. The most critical factors and their contribution to the prediction were identified using a modern tool of explainable artificial intelligence called SHapley Additive exPlanations (SHAP). RESULTS: The prediction model was based on an international cohort of 1184 patients and a validation cohort of 3543 patients. The best performing model was an XGBoost classifier with an average AUC score of 0.81 ± 0.033 and an accuracy of 89.1%, and the model improved with experience. The six most influential features were the respiratory rate, body temperature, abdominal muscular reflex, gender, age and glucose level. Using the XGBoost machine learning algorithm for prediction, the SHAP values for the explanation and the bootstrapping method to estimate confidence, we developed a free and easy-to-use web application in the Streamlit Python-based framework (http://easy-app.org/). CONCLUSIONS: The EASY prediction score is a practical tool for identifying patients at high risk for severe AP within hours of hospital admission. The web application is available for clinicians and contributes to the improvement of the model.
Asunto(s)
Inteligencia Artificial , Pancreatitis , Enfermedad Aguda , Humanos , Pancreatitis/diagnóstico , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
Acute pancreatitis (AP) is a severe and potentially fatal disease caused predominantly by alcohol excess and gallstones, which lacks a specific therapy. The role of Receptor-Interacting Protein Kinase 1 (RIPK1), a key component of programmed necrosis (Necroptosis), is unclear in AP. We assessed the effects of RIPK1 inhibitor Necrostatin-1 (Nec-1) and RIPK1 modification (RIPK1K45A: kinase dead) in bile acid (TLCS-AP), alcoholic (FAEE-AP) and caerulein hyperstimulation (CER-AP) mouse models. Involvement of collateral Nec-1 target indoleamine 2,3-dioxygenase (IDO) was probed with the inhibitor Epacadostat (EPA). Effects of Nec-1 and RIPK1K45A were also compared on pancreatic acinar cell (PAC) fate in vitro and underlying mechanisms explored. Nec-1 markedly ameliorated histological and biochemical changes in all models. However, these were only partially reduced or unchanged in RIPK1K45A mice. Inhibition of IDO with EPA was protective in TLCS-AP. Both Nec-1 and RIPK1K45A modification inhibited TLCS- and FAEE-induced PAC necrosis in vitro. Nec-1 did not affect TLCS-induced Ca2+ entry in PACs, however, it inhibited an associated ROS elevation. The results demonstrate protective actions of Nec-1 in multiple models. However, RIPK1-dependent necroptosis only partially contributed to beneficial effects, and actions on targets such as IDO are likely to be important.
Asunto(s)
Imidazoles/uso terapéutico , Indoles/uso terapéutico , Pancreatitis/tratamiento farmacológico , Pancreatitis/enzimología , Sustancias Protectoras/uso terapéutico , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Células Acinares/metabolismo , Alcoholes , Animales , Ácidos y Sales Biliares , Calcio/metabolismo , Ceruletida , Imidazoles/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Indoles/farmacología , Masculino , Ratones Endogámicos C57BL , Páncreas/patología , Pancreatitis/inducido químicamente , Sustancias Protectoras/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidoresRESUMEN
Acute pancreatitis is a frequent disease that lacks specific drug treatment. Unravelling the molecular mechanisms of acute pancreatitis is essential for the development of new therapeutics. Several inducers of acute pancreatitis trigger sustained Ca2+ increases in the cytosol and mitochondria of pancreatic acinar cells. The mitochondrial calcium uniporter (MCU) mediates mitochondrial Ca2+ uptake that regulates bioenergetics and plays an important role in cell survival, damage and death. Aberrant Ca2+ signaling and mitochondrial damage in pancreatic acinar cells have been implicated in the initiation of acute pancreatitis. The primary aim of this study was to assess the involvement of the MCU in experimental acute pancreatitis. We found that pancreatic acinar cells from MCU-/- mice display dramatically reduced mitochondrial Ca2+ uptake. This is consistent with the drastic changes of stimulus-metabolism coupling, manifested by the reduction of mitochondrial NADH/FAD+ responses to cholecystokinin and in the decrease of cholecystokinin-stimulated oxygen consumption. However, in three experimental models of acute pancreatitis (induced by caerulein, taurolithocholic acid 3-sulfate or palmitoleic acid plus ethanol), MCU knockout failed to reduce the biochemical and histological changes characterizing the severity of local and systemic damage. A possible explanation of this surprising finding is the redundancy of damaging mechanisms activated by the inducers of acute pancreatitis.
Asunto(s)
Células Acinares/metabolismo , Canales de Calcio/metabolismo , Páncreas/patología , Pancreatitis/metabolismo , Pancreatitis/patología , Índice de Severidad de la Enfermedad , Animales , Calcio/metabolismo , Señalización del Calcio , Citosol/metabolismo , Modelos Animales de Enfermedad , Flavina-Adenina Dinucleótido/metabolismo , Ratones Noqueados , Mitocondrias/metabolismo , NAD/metabolismoRESUMEN
Prion diseases are caused by accumulation of an abnormally folded isoform (PrP(Sc)) of the cellular prion protein (PrP(C)). The subcellular distribution of PrP(Sc) and the site of its formation in brain are still unclear. We performed quantitative cryo-immunogold electron microscopy on hippocampal sections from mice infected with the Rocky Mountain Laboratory strain of prions. Two antibodies were used: R2, which recognizes both PrP(C) and PrP(Sc); and F4-31, which only detects PrP(C) in undenatured sections. At a late subclinical stage of prion infection, both PrP(C) and PrP(Sc) were detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24-fold) was found on the early/recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrP(Sc) may be oligomeric, protease-sensitive PrP(Sc).
Asunto(s)
Microscopía por Crioelectrón/métodos , Proteínas PrPC/metabolismo , Proteínas PrPC/ultraestructura , Proteínas PrPSc/metabolismo , Proteínas PrPSc/ultraestructura , Animales , Dendritas/metabolismo , Dendritas/ultraestructura , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , Ratones , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Neurópilo/metabolismo , Proteínas PrPSc/genética , Enfermedades por Prión/etiología , Enfermedades por Prión/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
OBJECTIVES: Mitochondrial permeability transition pore inhibition is a promising approach to treat acute pancreatitis (AP). We sought to determine (i) the effects of the mitochondrial permeability transition pore inhibitor 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) on murine and human pancreatic acinar cell (PAC) injury induced by fatty acid ethyl esters (FAEEs) or taurolithocholic acid-3-sulfate and (ii) TRO40303 pharmacokinetics and efficacy in experimental alcoholic AP (FAEE-AP). METHODS: Changes in mitochondrial membrane potential (Δψm), cytosolic Ca ([Ca]c), and cell fate were examined in freshly isolated murine or human PACs by confocal microscopy. TRO40303 pharmacokinetics were assessed in cerulein-induced AP and therapeutic efficacy in FAEE-AP induced with palmitoleic acid and ethanol. Severity of AP was assessed by standard biomarkers and blinded histopathology. RESULTS: TRO40303 prevented loss of Δψm and necrosis induced by 100 µM palmitoleic acid ethyl ester or 500 µM taurolithocholic acid-3-sulfate in murine and human PACs. Pharmacokinetic analysis found TRO40303 accumulated in the pancreas. A single dose of 3 mg/kg TRO40303 significantly reduced serum amylase (P = 0.043), pancreatic trypsin (P = 0.018), and histopathology scores (P = 0.0058) in FAEE-AP. CONCLUSIONS: TRO40303 protects mitochondria and prevents necrotic cell death pathway activation in murine and human PACs, ameliorates the severity of FAEE-AP, and is a candidate drug for human AP.
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Ésteres/farmacología , Ácidos Grasos/farmacología , Mitocondrias/efectos de los fármacos , Oximas/farmacología , Pancreatitis Alcohólica/prevención & control , Secoesteroides/farmacología , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Enfermedad Aguda , Animales , Ceruletida , Ésteres/metabolismo , Ácidos Grasos/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Necrosis/prevención & control , Oximas/farmacocinética , Pancreatitis/inducido químicamente , Pancreatitis/prevención & control , Pancreatitis Alcohólica/metabolismo , Pancreatitis Alcohólica/patología , Secoesteroides/farmacocinética , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/farmacologíaRESUMEN
The insolubility of the disease-causing isoform of the prion protein (PrP(Sc)) has prevented studies of its three-dimensional structure at atomic resolution. Electron crystallography of two-dimensional crystals of N-terminally truncated PrP(Sc) (PrP 27-30) and a miniprion (PrP(Sc)106) provided the first insights at intermediate resolution on the molecular architecture of the prion. Here, we report on the structure of PrP 27-30 and PrP(Sc)106 negatively stained with heavy metals. The interactions of the heavy metals with the crystal lattice were governed by tertiary and quaternary structural elements of the protein as well as the charge and size of the heavy metal salts. Staining with molybdate anions revealed three prominent densities near the center of the trimer that forms the unit cell, coinciding with the location of the beta-helix that was proposed for the structure of PrP(Sc). Differential staining also confirmed the location of the internal deletion of PrP(Sc)106 at or near these densities.
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Cristalografía/métodos , Metales Pesados/química , Microscopía Electrónica/métodos , Modelos Moleculares , Proteínas PrPSc/química , Proteínas PrPSc/ultraestructura , Simulación por Computador , Interpretación de Imagen Asistida por Computador/métodos , Conformación ProteicaRESUMEN
Opening of the mitochondrial permeability transition pore (MPTP) causes mitochondrial dysfunction and necrosis in acute pancreatitis (AP), a condition without specific drug treatment. Cyclophilin D (CypD) is a mitochondrial matrix peptidyl-prolyl isomerase that regulates the MPTP and is a drug target for AP. We have synthesized urea-based small molecule inhibitors of cyclophilins and tested them against CypD using binding and isomerase activity assays. Thermodynamic profiles of the CypD/inhibitor interactions were determined by isothermal titration calorimetry. Seven new high-resolution crystal structures of CypD-inhibitor complexes were obtained to guide compound optimization. Compounds 4, 13, 14, and 19 were tested in freshly isolated murine pancreatic acinar cells (PACs) to determine inhibition of toxin-induced loss of mitochondrial membrane potential (ΔΨm) and necrotic cell death pathway activation. Compound 19 was found to have a Kd of 410 nM and a favorable thermodynamic profile, and it showed significant protection of ΔΨm and reduced necrosis of murine as well as human PACs. Compound 19 holds significant promise for future lead optimization.
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Ciclofilinas/antagonistas & inhibidores , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Pancreatitis Aguda Necrotizante/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Línea Celular , Cristalografía por Rayos X , Peptidil-Prolil Isomerasa F , Diseño de Fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Necrosis , Pirrolidinas/farmacología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , Termodinámica , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
BACKGROUND: Carboxyl-ester lipase (CEL) contributes to fatty acid ethyl ester metabolism, which is implicated in alcoholic pancreatitis. The CEL gene harbours a variable number of tandem repeats (VNTR) region in exon 11. Variation in this VNTR has been linked to monogenic pancreatic disease, while conflicting results were reported for chronic pancreatitis (CP). Here, we aimed to investigate a potential association of CEL VNTR lengths with alcoholic CP. METHODS: Overall, 395 alcoholic CP patients, 218 patients with alcoholic liver cirrhosis (ALC) serving as controls with a comparable amount of alcohol consumed, and 327 healthy controls from Germany and the United Kingdom (UK) were analysed by determination of fragment lengths by capillary electrophoresis. Allele frequencies and genotypes of different VNTR categories were compared between the groups. RESULTS: Twelve repeats were overrepresented in UK ACP patients (P = 0.04) compared to controls, whereas twelve repeats were enriched in German ALC compared to alcoholic CP patients (P = 0.03). Frequencies of CEL VNTR lengths of 14 and 15 repeats differed between German ALC patients and healthy controls (P = 0.03 and 0.008, respectively). However, in the genotype and pooled analysis of VNTR lengths no statistical significant association was depicted. Additionally, the 16-16 genotype as well as 16 repeats were more frequent in UK ALC than in alcoholic CP patients (P = 0.034 and 0.02, respectively). In all other calculations, including pooled German and UK data, allele frequencies and genotype distributions did not differ significantly between patients and controls or between alcoholic CP and ALC. CONCLUSIONS: We did not obtain evidence that CEL VNTR lengths are associated with alcoholic CP. However, our results suggest that CEL VNTR lengths might associate with ALC, a finding that needs to be clarified in larger cohorts.
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Lipasa/genética , Cirrosis Hepática Alcohólica/genética , Repeticiones de Minisatélite , Pancreatitis Alcohólica/genética , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad Crónica , Exones , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Alemania/epidemiología , Humanos , Cirrosis Hepática Alcohólica/epidemiología , Masculino , Persona de Mediana Edad , Pancreatitis Alcohólica/epidemiología , Reino Unido/epidemiologíaRESUMEN
Localizing the cellular prion protein (PrPC) in the brain is necessary for understanding the pathogenesis of prion diseases. However, the precise ultrastructural localization of PrPC still remains enigmatic. We performed the first quantitative study of the ultrastructural localization of PrPC in the mouse hippocampus using high-resolution cryoimmunogold electron microscopy. PrPC follows the standard biosynthetic trafficking pathway with a preferential localization in late endosomal compartments and on the plasma membrane of neurons and neuronal processes. PrPC is found with the same frequency within the synaptic specialization and perisynaptically, but is almost completely excluded from synaptic vesicles. Unexpectedly, PrP is also found in the cytosol in subpopulations of neurons in the hippocampus, neocortex, and thalamus but not the cerebellum. Cytosolic PrP may have altered susceptibility to aggregation, suggesting that these neurons might play a significant role in the pathogenesis of prion diseases, in particular those mammals harboring mutant PrP genes.
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Citosol/metabolismo , Neuronas/metabolismo , Proteínas PrPC/metabolismo , Animales , Especificidad de Anticuerpos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Cricetinae , Citosol/ultraestructura , Dendritas/metabolismo , Dendritas/ultraestructura , Endosomas/metabolismo , Endosomas/ultraestructura , Técnica del Anticuerpo Fluorescente , Hipocampo/metabolismo , Hipocampo/ultraestructura , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos , Ratones Transgénicos , Neuronas/ultraestructura , Neurópilo/metabolismo , Neurópilo/ultraestructura , Orgánulos/metabolismo , Orgánulos/ultraestructura , Proteínas PrPC/genética , Vesículas Sinápticas/ultraestructuraRESUMEN
In the present study, the in vivo distribution of polyelectrolyte multilayer coated gold nanoparticles is shown, starting from the living animal down to cellular level. The coating was designed with functional moieties to serve as a potential nano drug for prion disease. With near infrared time-domain imaging we followed the biodistribution in mice up to 7 days after intravenous injection of the nanoparticles. The peak concentration in the head of mice was detected between 19 and 24 h. The precise particle distribution in the brain was studied ex vivo by X-ray microtomography, confocal laser and fluorescence microscopy. We found that the particles mainly accumulate in the hippocampus, thalamus, hypothalamus, and the cerebral cortex.
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Encéfalo/metabolismo , Oro/química , Nanopartículas del Metal/química , Animales , Electrólitos/química , Humanos , Inyecciones Intravenosas , Nanopartículas del Metal/toxicidad , Ratones , Microscopía Fluorescente , Albúmina Sérica/química , Distribución Tisular , Microtomografía por Rayos XRESUMEN
The action of dopamine on the aggregation of the unstructured alpha-synuclein (alpha-syn) protein may be linked to the pathogenesis of Parkinson's disease. Dopamine and its oxidation derivatives may inhibit alpha-syn aggregation by non-covalent binding. Exploiting this fact, we applied an integrated computational and experimental approach to find alternative ligands that might modulate the fibrillization of alpha-syn. Ligands structurally and electrostatically similar to dopamine were screened from an established library. Five analogs were selected for in vitro experimentation from the similarity ranked list of analogs. Molecular dynamics simulations showed they were, like dopamine, binding non-covalently to alpha-syn and, although much weaker than dopamine, they shared some of its binding properties. In vitro fibrillization assays were performed on these five dopamine analogs. Consistent with our predictions, analyses by atomic force and transmission electron microscopy revealed that all of the selected ligands affected the aggregation process, albeit to a varying and lesser extent than dopamine, used as the control ligand. The in silico/in vitro approach presented here emerges as a possible strategy for identifying ligands interfering with such a complex process as the fibrillization of an unstructured protein.
Asunto(s)
Dopamina/análogos & derivados , Dopamina/química , alfa-Sinucleína/química , Dicroismo Circular , Dopamina/metabolismo , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/química , Indoles/metabolismo , Ligandos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , Estructura Molecular , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Electricidad Estática , Tiramina/química , Tiramina/metabolismo , Agua/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrP(Sc). The polyoxometalate phosphotungstic acid has been used to separate PrP(Sc) from its precursor PrP(C) by selective precipitation; notably, native PrP(Sc) has not been solubilized by using nondenaturing detergents. Because of the similarities between PrP(Sc) and lipoproteins with respect to hydrophobicity and formation of phosphotungstic acid complexes, we asked whether these molecules are bound to each other in blood. Here we report that prions from the brains of patients with sporadic Creutzfeldt-Jakob disease (CJD) bind to very low-density (VLDL) and low-density (LDL) lipoproteins but not to high-density lipoproteins (HDL) or other plasma components, as demonstrated both by affinity assay and electron microscopy. Immunoassays demonstrated that apolipoprotein B (apoB), which is the major protein component of VLDL and LDL, bound PrP(Sc) through a highly cooperative process. Approximately 50% of the PrP(Sc) bound to LDL particles was released after exposure to 4 M guanidine hydrochloride at 80 degrees C for 20 min. The apparent binding constants of native human (Hu) PrP(Sc) or denatured recombinant HuPrP(90-231) for apoB and LDL ranged from 28 to 212 pM. Whether detection of PrP(Sc) in VLDL and LDL particles can be adapted into an antemortem diagnostic test for prions in the blood of humans, livestock, and free-ranging cervids remains to be determined.