RESUMEN
The natural product curcumin and some of its analogs are known inhibitors of the transmembrane enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). Despite their widespread use, the curcuminoids' binding site in SERCA and their relevant interactions with the enzyme remain elusive. This lack of knowledge has prevented the development of curcuminoids into valuable experimental tools or into agents of therapeutic value. We used the crystal structures of SERCA in its E1 conformation in conjunction with computational tools such as docking and surface screens to determine the most likely curcumin binding site, along with key enzyme/inhibitor interactions. Additionally, we determined the inhibitory potencies and binding affinities for a small set of curcumin analogs. The predicted curcumin binding site is a narrow cleft in the transmembrane section of SERCA, close to the transmembrane/cytosol interface. In addition to pronounced complementarity in shape and hydrophobicity profiles between curcumin and the binding pocket, several hydrogen bonds were observed that were spread over the entire curcumin scaffold, involving residues on several transmembrane helices. Docking-predicted interactions were compatible with experimental observations for inhibitory potencies and binding affinities. Based on these findings, we propose an inhibition mechanism that assumes that the presence of a curcuminoid in the binding site arrests the catalytic cycle of SERCA by preventing it from converting from the E1 to the E2 conformation. This blockage of conformational change is accomplished by a combination of steric hinderance and hydrogen-bond-based cross-linking of transmembrane helices that require flexibility throughout the catalytic cycle.
Asunto(s)
Curcumina , Simulación del Acoplamiento Molecular , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Curcumina/química , Curcumina/farmacología , Curcumina/análogos & derivados , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Sitios de Unión , Animales , Enlace de Hidrógeno , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Unión ProteicaRESUMEN
Abnormal calcium signaling is associated with non-small cell lung cancer (NSCLC) malignant progression, poor survival and chemotherapy resistance. Targeting endoplasmic reticulum (ER) Ca2+ channels or pumps to block calcium uptake in the ER induces ER stress and concomitantly promotes mitochondrial calcium uptake, leading to mitochondrial dysfunction and ultimately inducing cell death. Here, we identified Diphyllin was a potential specific inhibitor of endoplasmic reticulum (ER) calcium-importing protein sarco/endoplasmic-reticulum Ca2+ ATPase 2 (SERCA2). In vitro and in vivo studies showed that Diphyllin increased NSCLC cell apoptosis, along with inhibition of cell proliferation and migration. Mechanistically, Diphyllin promoted ER stress by directly inhibiting SERCA2 activity and decreasing ER Ca2+ levels. At the same time, the accumulated Ca2+ in cytoplasm flowed into mitochondria to increase reactive oxygen species (ROS) and decrease mitochondrial membrane potential (MMP), leading to cytochrome C (Cyto C) release and mitochondrial dysfunction. In addition, we found that Diphyllin combined with cisplatin could have a synergistic anti-tumor effect in vitro and in vivo. Taken together, our results suggested that Diphyllin, as a potential novel inhibitor of SERCA2, exerts anti-tumor effects by blocking ER Ca2+ uptake and thereby promoting ER stress and mitochondrial dysfunction.
Asunto(s)
Apoptosis , Carcinoma de Pulmón de Células no Pequeñas , Proliferación Celular , Estrés del Retículo Endoplásmico , Neoplasias Pulmonares , Mitocondrias , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Estrés del Retículo Endoplásmico/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Animales , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ratones , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Calcio/metabolismo , Células A549 , Sinergismo Farmacológico , Ratones Desnudos , Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Cisplatino/farmacología , Ratones Endogámicos BALB C , Señalización del Calcio/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismoRESUMEN
We explored the vasorelaxant effects of ipragliflozin, a sodium-glucose cotransporter-2 inhibitor, on rabbit femoral arterial rings. Ipragliflozin relaxed phenylephrine-induced pre-contracted rings in a dose-dependent manner. Pre-treatment with the ATP-sensitive K+ channel inhibitor glibenclamide (10 µM), the inwardly rectifying K+ channel inhibitor Ba2+ (50 µM), or the Ca2+-sensitive K+ channel inhibitor paxilline (10 µM) did not influence the vasorelaxant effect. However, the voltage-dependent K+ (Kv) channel inhibitor 4-aminopyridine (3 mM) reduced the vasorelaxant effect. Specifically, the vasorelaxant response to ipragliflozin was significantly attenuated by pretreatment with the Kv7.X channel inhibitors linopirdine (10 µM) and XE991 (10 µM), the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin (1 µM) and cyclopiazonic acid (10 µM), and the cAMP/protein kinase A (PKA)-associated signaling pathway inhibitors SQ22536 (50 µM) and KT5720 (1 µM). Neither the cGMP/protein kinase G (PKG)-associated signaling pathway nor the endothelium was involved in ipragliflozin-induced vasorelaxation. We conclude that ipragliflozin induced vasorelaxation of rabbit femoral arteries by activating Kv channels (principally the Kv7.X channel), the SERCA pump, and the cAMP/PKA-associated signaling pathway independent of other K+ (ATP-sensitive K+, inwardly rectifying K+, and Ca2+-sensitive K+) channels, cGMP/PKG-associated signaling, and the endothelium.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico , Arteria Femoral , Glucósidos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Transducción de Señal , Tiofenos , Vasodilatación , Animales , Conejos , Arteria Femoral/efectos de los fármacos , Arteria Femoral/fisiología , Vasodilatación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Tiofenos/farmacología , Masculino , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Vasodilatadores/farmacología , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidoresRESUMEN
BACKGROUD: Endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is crucial for maintaining intracellular calcium homeostasis due to the calcium-signaling-related proteins on its membrane. While ryanodine receptors (RyR) on insect ER/SR membranes are well-known as targets for diamide insecticides, little is known about other calcium channels. Given the resistance of diamide insecticides, the establishment of molecular screening models targeting RyR or sarco/endoplasmic reticulum calcium ATPase (SERCA) is conducive to the discovery of new insecticidal molecules. RESULTS: The morphological features of Mythimna separata SR have closed vesicles with integrity and high density. The 282 proteins in the SR component contained RyR and SERCA. A measurement model for the release and uptake of calcium was successfully established by detecting calcium ions outside the SR membrane using a fluorescence spectrophotometer. In vitro testing systems using SR vesicles found that diamide insecticides could activate dose-dependently RyR, with EC50 values of 0.14 µM (Chlorantraniliprole), 0.21 µM (Flubendiamide), and 0.57 µM (Cyantraniliprole), respectively. However, dantrolene inhibited RyR-mediated calcium release with an IC50 value of 353.9 µM, suggesting that dantrolene can weakly antagonize RyR. Moreover, cyclopiazonic acid significantly reduced the enzyme activity and calcium uptake capacity of SERCA. On the contrary, CDN1163 markedly activated the enzyme activity and improved the calcium transport capacity of SERCA. CONCLUSIONS: SR vesicles can be used to study the function of unknown proteins on the SR membranes, as well as for high-throughput screening of highly active compounds targeting RyR or SERCA. © 2024 Society of Chemical Industry.
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Insecticidas/farmacología , Retículo Sarcoplasmático/metabolismo , Calcio/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Retículo Endoplásmico/metabolismoRESUMEN
We report a novel small-molecule screening approach that combines data augmentation and machine learning to identify Food and Drug Administration (FDA)-approved drugs interacting with the calcium pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA) from skeletal (SERCA1a) and cardiac (SERCA2a) muscle. This approach uses information about small-molecule effectors to map and probe the chemical space of pharmacological targets, thus allowing to screen with high precision large databases of small molecules, including approved and investigational drugs. We chose SERCA because it plays a major role in the excitation-contraction-relaxation cycle in muscle and it represents a major target in both skeletal and cardiac muscle. The machine learning model predicted that SERCA1a and SERCA2a are pharmacological targets for seven statins, a group of FDA-approved 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used in the clinic as lipid-lowering medications. We validated the machine learning predictions by using in vitro ATPase assays to show that several FDA-approved statins are partial inhibitors of SERCA1a and SERCA2a. Complementary atomistic simulations predict that these drugs bind to two different allosteric sites of the pump. Our findings suggest that SERCA-mediated Ca2+ transport may be targeted by some statins (e.g., atorvastatin), thus providing a molecular pathway to explain statin-associated toxicity reported in the literature. These studies show the applicability of data augmentation and machine learning-based screening as a general platform for the identification of off-target interactions and the applicability of this approach extends to drug discovery.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Miocardio/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Aprendizaje AutomáticoRESUMEN
The detailed analysis and structural characterization of proteoforms by top-down proteomics (TDP) has gained a lot of interest in biomedical research. Data-dependent acquisition (DDA) of intact proteins is non-trivial due to the diversity and complexity of proteoforms. Dedicated acquisition methods thus have the potential to greatly improve TDP. Here, we present FLASHIda, an intelligent online data acquisition algorithm for TDP that ensures the real-time selection of high-quality precursors of diverse proteoforms. FLASHIda combines fast charge deconvolution algorithms and machine learning-based quality assessment for optimal precursor selection. In an analysis of E. coli lysate, FLASHIda increases the number of unique proteoform level identifications from 800 to 1500 or generates a near-identical number of identifications in one third of the instrument time when compared to standard DDA mode. Furthermore, FLASHIda enables sensitive mapping of post-translational modifications and detection of chemical adducts. As a software extension module to the instrument, FLASHIda can be readily adopted for TDP studies of complex samples to enhance proteoform identification rates.
Asunto(s)
Proteoma , Proteómica , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Corazón , Péptidos , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is a crucial component of the cellular machinery responsible for Ca2+ homeostasis. The selective inhibition of SERCA by thapsigargin (TG) leads to perturbations in Ca2+ signaling, which can trigger endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) pathway is activated in response to ER stress and induces an adaptive response to preserve cell survival or committee cells to programmed death, depending on stress duration and/or level. Early stages of ER stress stimulate mitochondrial metabolism to preserve survival but under chronic ER stress conditions, mitochondrial dysfunction is induced, which, in turn, can enhance inflammation through NLRP3 inflammasome activation. This study was aimed at investigating the role of SERCA inhibition on NLRP3 inflammasome activation in human monocytes, which was evaluated in primary monocytes isolated from healthy individuals and in the THP-1 human monocytic cell line. Findings obtained in both THP-1 and primary monocytes demonstrate that SERCA inhibition triggered by TG does not activate the NLRP3 inflammasome in these innate immune cells since IL-1ß secretion was not affected. Results from THP-1 monocytes showing that SERCA inhibition increases mitochondrial Ca2+ content and fusion, in the absence of changes in ROS levels and membrane potential, support the view that human monocytes counteract ER stress that arises from inhibition of SERCA through modulation of mitochondrial morphology towards mitochondria fusion, thus preventing NLRP3 inflammasome activation. Overall, this work contributes to a better understanding of the molecular mechanisms that modulate the activity of the NLRP3 inflammasome leading to sterile inflammation, which are still poorly understood.
Asunto(s)
Inflamasomas , Dinámicas Mitocondriales , Proteína con Dominio Pirina 3 de la Familia NLR , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Humanos , Inflamasomas/metabolismo , Inflamación/metabolismo , Mitocondrias/metabolismo , Monocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Tapsigargina/farmacologíaRESUMEN
Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na+ and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na+-permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca2+ release and turned off by Ca2+ reuptake through the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output.
Asunto(s)
Locomoción/fisiología , Neuronas Motoras/metabolismo , Paresia/fisiopatología , Médula Espinal/fisiología , Canales Catiónicos TRPM/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Simulación por Computador , Modelos Animales de Enfermedad , Femenino , Silenciador del Gen , Células HEK293 , Miembro Posterior/fisiología , Humanos , Locomoción/efectos de los fármacos , Masculino , Ratones , Neuronas Motoras/efectos de los fármacos , Paresia/genética , Técnicas de Placa-Clamp , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Médula Espinal/citología , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/genéticaRESUMEN
A series of eleven celastrol derivatives was designed, synthesized, and evaluated for their in vitro cytotoxic activities against six human cancer cell lines (A549, HepG2, HepAD38, PC3, DLD-1 Bax-Bak WT and DKO) and three human normal cells (LO2, BEAS-2B, CCD19Lu). To our knowledge, six derivatives were the first example of dipeptide celastrol derivatives. Among them, compound 3 was the most promising derivative, as it exhibited a remarkable anti-proliferative activity and improved selectivity in liver cancer HepAD38 versus human normal hepatocytes, LO2. Compound 6 showed higher selectivity in liver cancer cells against human normal lung fibroblasts, CCD19Lu cell line. The Ca2+ mobilizations of 3 and 6 were also evaluated in the presence and absence of thapsigargin to demonstrate their inhibitory effects on SERCA. Derivatives 3 and 6 were found to induce apoptosis on LO2, HepG2 and HepAD38 cells. The potential docking poses of all synthesized celastrol dipeptides and other known inhibitors were proposed by molecular docking. Finally, 3 inhibited P-gp-mediated drug efflux with greater efficiency than inhibitor verapamil in A549 lung cancer cells. Therefore, celastrol-dipeptide derivatives are potent drug candidates for the treatment of drug-resistant cancer.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/síntesis química , Simulación del Acoplamiento Molecular , Triterpenos Pentacíclicos/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Triterpenos Pentacíclicos/metabolismo , Triterpenos Pentacíclicos/farmacología , Triterpenos Pentacíclicos/uso terapéutico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Relación Estructura-ActividadRESUMEN
We have reported recently that submaximal inhibition of the Sarco Endoplasmic Reticulum Ca2+ ATPase (SERCA) produces an increase in the lifespan of C. elegans worms. We have explored here the mechanism of this increased survival by studying the effect of SERCA inhibition in several mutants of signaling pathways related to longevity. Our data show that the mechanism of the effect is unrelated with the insulin signaling pathway or the sirtuin activity, because SERCA inhibitors increased lifespan similarly in mutants of these pathways. However, the effect required functional mitochondria and both the AMP kinase and TOR pathways, as the SERCA inhibitors were ineffective in the corresponding mutants. The same effects were obtained after reducing SERCA expression with submaximal RNAi treatment. The SERCA inhibitors did not induce ER-stress at the concentrations used, and their effect was not modified by inactivation of the OP50 bacterial food. Altogether, our data suggest that the effect may be due to a reduced ER-mitochondria Ca2+ transfer acting via AMPK activation and mTOR inhibition to promote survival.
Asunto(s)
Retículo Endoplásmico , Insulina/metabolismo , Longevidad , Mitocondrias/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tapsigargina/farmacología , Proteínas Quinasas Activadas por AMP , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/genética , Longevidad/efectos de los fármacos , Longevidad/fisiología , Tratamiento con ARN de Interferencia/métodos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sirtuinas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Resultado del TratamientoRESUMEN
Skeletal muscle Na+ channels possess Ca2+- and calmodulin-binding sites implicated in Nav1.4 current (INa) downregulation following ryanodine receptor (RyR1) activation produced by exchange protein directly activated by cyclic AMP or caffeine challenge, effects abrogated by the RyR1-antagonist dantrolene which itself increased INa. These findings were attributed to actions of consequently altered cytosolic Ca2+, [Ca2+]i, on Nav1.4. We extend the latter hypothesis employing cyclopiazonic acid (CPA) challenge, which similarly increases [Ca2+]i, but through contrastingly inhibiting sarcoplasmic reticular (SR) Ca2+-ATPase. Loose patch clamping determined Na+ current (INa) families in intact native murine gastrocnemius skeletal myocytes, minimising artefactual [Ca2+]i perturbations. A bespoke flow system permitted continuous INa comparisons through graded depolarizing steps in identical stable membrane patches before and following solution change. In contrast to the previous studies modifying RyR1 activity, and imposing control solution changes, CPA (0.1 and 1 µM) produced persistent increases in INa within 1-4 min of introduction. CPA pre-treatment additionally abrogated previously reported reductions in INa produced by 0.5 mM caffeine. Plots of peak current against voltage excursion demonstrated that 1 µM CPA increased maximum INa by ~ 30%. It only slightly decreased half-maximal activating voltages (V0.5) and steepness factors (k), by 2 mV and 0.7, in contrast to the V0.5 and k shifts reported with direct RyR1 modification. These paradoxical findings complement previously reported downregulatory effects on Nav1.4 of RyR1-agonist mediated increases in bulk cytosolic [Ca2+]. They implicate possible local tubule-sarcoplasmic triadic domains containing reduced [Ca2+]TSR in the observed upregulation of Nav1.4 function following CPA-induced SR Ca2+ depletion.
Asunto(s)
Músculo Esquelético/metabolismo , Canal de Sodio Activado por Voltaje NAV1.4/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Cafeína/farmacología , Agonistas de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Indoles/farmacología , Ratones , Fibras Musculares Esqueléticas , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Técnicas de Placa-Clamp , Cultivo Primario de Células , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Sodio/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with calcium ATPase from sarcoplasmic reticulum (SR) was studied by means of different approaches, such as enzyme activity, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and molecular docking simulations. The ATP hydrolysis activity was fully inhibited by incubation with dirhamnolipid (diRL) up to 0.1 mM concentration, corresponding to a surfactant concentration below membrane solubilization threshold. Surfactant-protein interaction induced conformational changes in the protein observed by an increase in the accessibility of tryptophan residues to the aqueous phase and by changes in the secondary structure of the protein as seen by fluorescence and FTIR spectroscopy. As a consequence, the protein become more unstable and denatured at lower temperatures, as seen by enzyme activity and DSC studies. Finally, these results were explained at molecular level throughout molecular docking simulations. It is concluded that there is a specific dirhamnolipid-protein interaction not related to the surface activity of the surfactant but to the particular physicochemical properties of the biosurfactant molecule.
Asunto(s)
Glucolípidos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tensoactivos/metabolismo , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Glucolípidos/química , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Secundaria de Proteína/efectos de los fármacos , Pseudomonas aeruginosa/química , Conejos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , Tensoactivos/químicaRESUMEN
The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). Given that FAs are the components of membranes, FA metabolic enzymes might be associated with regulation of ER membrane functions. However, it remains unclear whether there is the interplay between FA metabolic enzymes and ER membrane proteins. Trans-2-enoyl-CoA reductase (TER) is an FA reductase present in the ER membrane and catalyzes the last step in the FA elongation cycle and sphingosine degradation pathway. Here we identify sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), an ER Ca2+ pump responsible for Ca2+ accumulation in the ER, as a TER-binding protein by affinity purification from HEK293 cell lysates. We show that TER directly binds to SERCA2b by in vitro assays using recombinant proteins. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes of HEK293 cells. Depletion of TER increases Ca2+ storage in the ER and accelerates SERCA2b-dependent Ca2+ uptake to the ER after ligand-induced Ca2+ release. Moreover, depletion of TER reduces the Ca2+-dependent nuclear translocation of nuclear factor of activated T cells 4. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca2+ accumulation in the ER.
Asunto(s)
Retículo Endoplásmico/metabolismo , Ácidos Grasos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Transporte Activo de Núcleo Celular/genética , Calcio/metabolismo , Señalización del Calcio/genética , Retículo Endoplásmico/genética , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Ligandos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Unión Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/químicaRESUMEN
P-type ATPase inhibitors are among the most successful and widely prescribed therapeutics in modern pharmacology. Clinical transition has been safely achieved for H+/K+ ATPase inhibitors such as omeprazole and Na+/K+-ATPase inhibitors like digoxin. However, this is more challenging for Ca2+-ATPase modulators due to the physiological role of Ca2+ in cardiac dynamics. Over the past two decades, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) modulators have been studied as potential chemotherapy agents because of their Ca2+-mediated pan-cancer lethal effects. Instead, recent evidence suggests that SERCA inhibition suppresses oncogenic Notch1 signaling emerging as an alternative to γ-secretase modulators that showed limited clinical activity due to severe side effects. In this review, we focus on how SERCA inhibitors alter Notch1 signaling and show that Notch on-target-mediated antileukemia properties of these molecules can be achieved without causing overt Ca2+ cellular overload.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Receptores Notch/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Terapia Molecular Dirigida , Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores Notch/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tapsigargina/análogos & derivados , Tapsigargina/farmacología , Tapsigargina/uso terapéuticoRESUMEN
Pre-blockade of the sarco-endoplasmic reticulum (ER) calcium ATPase (SERCA) with irreversible thapsigargin depresses exocytosis in adrenal bovine chromaffin cells (BCCs). Distinct expression of voltage-dependent Ca2+-channel subtypes and of the Ca2+-induced Ca2+ release (CICR) mechanism in BCCs versus mouse chromaffin cells (MCCs) has been described. We present a parallel study on the effects of the acute SERCA blockade with reversible cyclopizonic acid (CPA), to repeated pulsing with acetylcholine (ACh) at short (15 s) and long intervals (60 s) at 37 °C, allowing the monitoring of the initial size of a ready-release vesicle pool (RRP) and its depletion and recovery in subsequent stimuli. We found (i) strong depression of exocytosis upon ACh pulsing at 15-s intervals and slower depression at 60-s intervals in both cell types; (ii) facilitation of exocytosis upon acute SERCA inhibition, with back to depression upon CPA washout in MCCs; (iii) blockade of exocytosis upon acute SERCA inhibition and pronounced rebound of exocytosis upon CPA washout in BCCs; (iv) basal [Ca2+]c elevation upon stimulation with ACh at short intervals (but not at long intervals) in both cell types; and (v) augmentation of basal [Ca2+]c and inhibition of peak [Ca2+]c amplitude upon CPA treatment in both cell types, with milder effects upon stimulation at 60-s intervals. These results are compatible with the view that while in MCCs the uptake of Ca2+ via SERCA contributes to the mitigation of physiological ACh triggered secretion, in BCCs the uptake of Ca2+ into the ER facilitates such responses likely potentiating a Ca2+-induced Ca2+ release mechanism. These drastic differences in the regulation of ACh-triggered secretion at 37 °C may help to understand different patterns of the regulation of exocytosis by the circulation of Ca2+ at a functional ER Ca2+ store.
Asunto(s)
Calcio/metabolismo , Catecolaminas/metabolismo , Células Cromafines/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Exocitosis/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Acetilcolina/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Bovinos , Células Cultivadas , Células Cromafines/enzimología , Retículo Endoplásmico/enzimología , Indoles/farmacología , Masculino , Ratones Endogámicos C57BL , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Especificidad de la Especie , Tapsigargina/farmacología , Factores de TiempoRESUMEN
In spite of the impressing cytotoxicity of thapsigargin (Tg), this compound cannot be used as a chemotherapeutic drug because of general toxicity, causing unacceptable side effects. Instead, a prodrug targeted towards tumors, mipsagargin, was brought into clinical trials. What substantially reduces the clinical potential is the limited access to Tg and its derivatives and cost-inefficient syntheses with unacceptably low yields. Laser trilobum, which contains a structurally related sesquiterpene lactone, trilobolide (Tb), is successfully cultivated. Here, we report scalable isolation of Tb from L. trilobum and a transformation of Tb to 8-O-(12-aminododecanoyl)-8-O-debutanoylthapsigargin in seven steps. The use of cultivated L. trilobum offers an unlimited source of the active principle in mipsagargin.
Asunto(s)
Antineoplásicos Fitogénicos/química , Apiaceae/química , Butiratos/química , Técnicas de Química Sintética , Furanos/química , Tapsigargina/análogos & derivados , Antineoplásicos Fitogénicos/aislamiento & purificación , Apiaceae/metabolismo , Butiratos/aislamiento & purificación , Dióxido de Carbono/química , Cromatografía con Fluido Supercrítico/métodos , Frutas/química , Frutas/metabolismo , Furanos/aislamiento & purificación , Humanos , Estructura Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Extractos Vegetales/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tapsigargina/aislamiento & purificaciónRESUMEN
Despite its outstanding clinical success, immune checkpoint blockade remains ineffective in many patients. Accordingly, combination therapy capable of achieving greater antitumor immunity is urgently required. Here, we report that limiting glutamine metabolism in cancer cells bolsters the effectiveness of anti-programmed death ligand-1 (PD-L1) antibody. Inhibition of glutamine utilization increased PD-L1 levels in cancer cells, thereby inactivating co-cultured T cells. Under glutamine-limited conditions, reduced cellular GSH levels caused an upregulation of PD-L1 expression by impairing SERCA activity, which activates the calcium/NF-κB signaling cascade. Consequently, in tumors grown in immunocompetent mice, inhibition of glutamine metabolism decreased the antitumor activity of T cells. In combination with anti-PD-L1, however, glutamine depletion strongly promoted the antitumor efficacy of T cells in vitro and in vivo due to simultaneous increases in Fas/CD95 levels. Our results demonstrate the relevance of cancer glutamine metabolism to antitumor immunity and suggest that co-targeting of glutamine metabolism and PD-L1 represents a promising therapeutic approach.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno B7-H1/metabolismo , Glutamina/metabolismo , Glutatión/metabolismo , Neoplasias/inmunología , Neoplasias/prevención & control , Linfocitos T/inmunología , Anciano , Animales , Apoptosis , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Proliferación Celular , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/patología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Viral infection is one environmental factor that may contribute to the initiation of pancreatic ß-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate ß-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the ß-cell response to viral infection. We show that nitric oxide protects ß-cells against virally mediated lysis by limiting EMCV replication. This protection requires low micromolar, or iNOS-derived, levels of nitric oxide. At these concentrations nitric oxide inhibits the Krebs enzyme aconitase and complex IV of the electron transport chain. Like nitric oxide, pharmacological inhibition of mitochondrial oxidative metabolism attenuates EMCV-mediated ß-cell lysis by inhibiting viral replication. These findings provide novel evidence that cytokine signaling in ß-cells functions to limit viral replication and subsequent ß-cell lysis by attenuating mitochondrial oxidative metabolism in a nitric oxide-dependent manner.
Asunto(s)
Virus de la Encefalomiocarditis/fisiología , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Femenino , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/virología , Interferón beta/genética , Interferón beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/farmacología , Estrés Oxidativo/efectos de los fármacos , Poli I-C/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Replicación ViralRESUMEN
Influenza A virus is a major global pathogen of humans, and there is an unmet need for effective antivirals. Current antivirals against influenza A virus directly target the virus and are vulnerable to mutational resistance. Harnessing an effective host antiviral response is an attractive alternative. We show that brief exposure to low, non-toxic doses of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, promptly elicits an extended antiviral state that dramatically blocks influenza A virus production. Crucially, oral administration of TG protected mice against lethal virus infection and reduced virus titres in the lungs of treated mice. TG-induced ER stress unfolded protein response appears as a key driver responsible for activating a spectrum of host antiviral defences that include an enhanced type I/III interferon response. Our findings suggest that TG is potentially a viable host-centric antiviral for the treatment of influenza A virus infection without the inherent problem of drug resistance.
Asunto(s)
Antivirales/farmacología , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H3N8 del Virus de la Influenza A/crecimiento & desarrollo , Tapsigargina/farmacología , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , Perros , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Gripe Humana/tratamiento farmacológico , Interferón Tipo I/efectos de los fármacos , Interferón Tipo I/inmunología , Interferones/efectos de los fármacos , Interferones/inmunología , Ratones , Ratones Endogámicos BALB C , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Porcinos , Respuesta de Proteína Desplegada/efectos de los fármacos , Células Vero , Interferón lambdaRESUMEN
Spontaneous Ca2+-transient (wave) generation in isolated cardiomyocytes is well established phenomenon which poses a lot of questions about myocardial excitability. Current studies of spontaneous Ca2+-activity in cardiac cells mainly relate to the kinetic characteristics, classification and simulation of Ca2+-events through ryanodine receptor (RyR) activity modeling. Here, for the first time we pay attention to the Ca2+-transients having stationary kinetics for correct estimation of the sarcoplasmic reticulum Ca2+ transport. In cardiomyocytes generating such type of Ca2+-transients, the averaged intracellular calcium ([Ca2+]in) fluorescence practically does not change in time. Stationary Ca2+-transients are observed in different animal models (Wistar, SHR, ground squirrels) revealing a common cardiomyocyte phenomenon. They somewhat depend on external Ca2+ ([Ca2+]ex) because the [Ca2+]ex lowering to 1 µM in the presence of EGTA disrupts Ca2+-wave propagation. At the same time, spontaneous Ca2+-transients do not associated with the forward or reverse mode of Na+/Ca2+ exchanger (NCX), but partially modulated by the L-type Ca2+-channels. Among the sarcoplasmic reticulum targets, RyR and SERCA are crucial for Ca2+-wave generation and sustained self-oscillation activity. Analysis of the spontaneous wave kinetics reveals that both slopes of the rising wave front and the wave front decline are gradually changed during propagation, which well correlates with the RyR and SERCA activity, respectively. On the contrary, in the electrical field-stimulated myocytes, both slope factors are sharply changed corresponding to 'all-or-nothing' rule, which is fundamental principle for action potential in cardiomyocytes. Furthermore, stimulation of single cardiomyocyte using local electrode appears the deterioration in the [Ca2+]in utilization from the cytosol, which limits the time of observation during the protocol. Obtained data suggest that stationary spontaneous Ca2+-transients occurring without actual myocellular excitation represent useful and precise tools for estimation of the sarcoplasmic reticulum Ca2+-transport.