Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 103
Filtrar
1.
Sci Signal ; 17(842): eadi0934, 2024 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-38917219

RESUMEN

The stabilization of different active conformations of G protein-coupled receptors is thought to underlie the varying efficacies of biased and balanced agonists. Here, profiling the activation of signal transducers by angiotensin II type 1 receptor (AT1R) agonists revealed that the extent and kinetics of ß-arrestin binding exhibited substantial ligand-dependent differences, which were lost when receptor internalization was inhibited. When AT1R endocytosis was prevented, even weak partial agonists of the ß-arrestin pathway acted as full or near-full agonists, suggesting that receptor conformation did not exclusively determine ß-arrestin recruitment. The ligand-dependent variance in ß-arrestin translocation was much larger at endosomes than at the plasma membrane, showing that ligand efficacy in the ß-arrestin pathway was spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shaped the effects of agonists on endosomal receptor-ß-arrestin binding and thus determined the extent of functional selectivity. Ligand dissociation rate and G protein activity had particularly strong, internalization-dependent effects on the receptor-ß-arrestin interaction. We also showed that endocytosis regulated the agonist efficacies of two other receptors with sustained ß-arrestin binding: the V2 vasopressin receptor and a mutant ß2-adrenergic receptor. In the absence of endocytosis, the agonist-dependent variance in ß-arrestin2 binding was markedly diminished. Our results suggest that endocytosis determines the spatiotemporal bias in GPCR signaling and can aid in the development of more efficacious, functionally selective compounds.


Asunto(s)
Endocitosis , Receptor de Angiotensina Tipo 1 , Transducción de Señal , beta-Arrestinas , Endocitosis/fisiología , Humanos , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 1/genética , beta-Arrestinas/metabolismo , beta-Arrestinas/genética , Células HEK293 , Receptores de Vasopresinas/metabolismo , Receptores de Vasopresinas/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/genética , Endosomas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Animales , Ligandos , Unión Proteica , Transporte de Proteínas
2.
Cell Rep ; 43(5): 114241, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38758647

RESUMEN

The binding and function of ß-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. We employ a 1D sequence convolution model trained on GPCRs with established ß-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with ß-arrestins can be identified, a pattern that we name "arreSTick." Intriguingly, the arreSTick pattern is also present in numerous non-receptor proteins. Using proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between many non-receptor proteins and ß-arrestin2. The HIV-1 Tat-specific factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and its subcellular localization is influenced by ß-arrestin2. Our findings unveil a broader role for ß-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.


Asunto(s)
Secuencias de Aminoácidos , Unión Proteica , beta-Arrestinas , Fosforilación , Humanos , beta-Arrestinas/metabolismo , Células HEK293 , Arrestina beta 2/metabolismo , Secuencia de Aminoácidos , Estabilidad Proteica
3.
Contact (Thousand Oaks) ; 7: 25152564241229273, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38362008

RESUMEN

Calcium signal propagation from endoplasmic reticulum (ER) to mitochondria regulates a multitude of mitochondrial and cell functions, including oxidative ATP production and cell fate decisions. Ca2+ transfer is optimal at the ER-mitochondrial contacts, where inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) can locally expose the mitochondrial Ca2+ uniporter (mtCU) to high [Ca2+] nanodomains. The Ca2+ loading state of the ER (Ca2 + ER) can vary broadly in physiological and pathological scenarios, however, the correlation between Ca2 + ER and the local Ca2+ transfer is unclear. Here, we studied IP3-induced Ca2+ transfer to mitochondria at different Ca2 + ER in intact and permeabilized RBL-2H3 cells via fluorescence measurements of cytoplasmic [Ca2+] ([Ca2+]c) and mitochondrial matrix [Ca2+] ([Ca2+]m). Preincubation of intact cells in high versus low extracellular [Ca2+] caused disproportionally greater increase in [Ca2+]m than [Ca2+]c responses to IP3-mobilizing agonist. Increasing Ca2 + ER by small Ca2+ boluses in suspensions of permeabilized cells supralinearly enhanced the mitochondrial Ca2+ uptake from IP3-induced Ca2+ release. The IP3-induced local [Ca2+] spikes exposing the mitochondrial surface measured using a genetically targeted sensor appeared to linearly correlate with Ca2 + ER, indicating that amplification happened in the mitochondria. Indeed, overexpression of an EF-hand deficient mutant of the mtCU gatekeeper MICU1 reduced the cooperativity of mitochondrial Ca2+ uptake. Interestingly, the IP3-induced [Ca2+]m signal plateaued at high Ca2 + ER, indicating activation of a matrix Ca2+ binding/chelating species. Mitochondria thus seem to maintain a "working [Ca2+]m range" via a low-affinity and high-capacity buffer species, and the ER loading steeply enhances the IP3R-linked [Ca2+]m signals in this working range.

4.
Sci Rep ; 14(1): 291, 2024 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-38168911

RESUMEN

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the ß2 adrenergic receptor (ß2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on ß2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of ß2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, ß2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of ß2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease ß2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.


Asunto(s)
Endocitosis , Fosfatidilinositoles , Fosfatidilinositoles/metabolismo , Membrana Celular/metabolismo , Receptores de Angiotensina/metabolismo , Hormonas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo
6.
Circ Res ; 132(11): e171-e187, 2023 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-37057625

RESUMEN

BACKGROUND: Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering. METHODS: We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype. RESULTS: Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive ß-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury. CONCLUSIONS: We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.


Asunto(s)
Señalización del Calcio , Retículo Sarcoplasmático , Femenino , Ratones , Animales , Retículo Sarcoplasmático/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Mitocondrias Cardíacas/metabolismo , Calcio/metabolismo
7.
Int J Mol Sci ; 24(4)2023 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-36835391

RESUMEN

Angiotensin II (AngII) is a vasoactive peptide hormone, which, under pathological conditions, contributes to the development of cardiovascular diseases. Oxysterols, including 25-hydroxycholesterol (25-HC), the product of cholesterol-25-hydroxylase (CH25H), also have detrimental effects on vascular health by affecting vascular smooth muscle cells (VSMCs). We investigated AngII-induced gene expression changes in VSMCs to explore whether AngII stimulus and 25-HC production have a connection in the vasculature. RNA-sequencing revealed that Ch25h is significantly upregulated in response to AngII stimulus. The Ch25h mRNA levels were elevated robustly (~50-fold) 1 h after AngII (100 nM) stimulation compared to baseline levels. Using inhibitors, we specified that the AngII-induced Ch25h upregulation is type 1 angiotensin II receptor- and Gq/11 activity-dependent. Furthermore, p38 MAPK has a crucial role in the upregulation of Ch25h. We performed LC-MS/MS to identify 25-HC in the supernatant of AngII-stimulated VSMCs. In the supernatants, 25-HC concentration peaked 4 h after AngII stimulation. Our findings provide insight into the pathways mediating AngII-induced Ch25h upregulation. Our study elucidates a connection between AngII stimulus and 25-HC production in primary rat VSMCs. These results potentially lead to the identification and understanding of new mechanisms in the pathogenesis of vascular impairments.


Asunto(s)
Angiotensina II , Músculo Liso Vascular , Esteroide Hidroxilasas , Animales , Ratas , Angiotensina II/metabolismo , Células Cultivadas , Cromatografía Liquida , Expresión Génica , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/metabolismo , Espectrometría de Masas en Tándem , Esteroide Hidroxilasas/genética
8.
Nat Commun ; 13(1): 6779, 2022 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-36351901

RESUMEN

Endoplasmic reticulum-mitochondria contacts (ERMCs) are restructured in response to changes in cell state. While this restructuring has been implicated as a cause or consequence of pathology in numerous systems, the underlying molecular dynamics are poorly understood. Here, we show means to visualize the capture of motile IP3 receptors (IP3Rs) at ERMCs and document the immediate consequences for calcium signaling and metabolism. IP3Rs are of particular interest because their presence provides a scaffold for ERMCs that mediate local calcium signaling, and their function outside of ERMCs depends on their motility. Unexpectedly, in a cell model with little ERMC Ca2+ coupling, IP3Rs captured at mitochondria promptly mediate Ca2+ transfer, stimulating mitochondrial oxidative metabolism. The Ca2+ transfer does not require linkage with a pore-forming protein in the outer mitochondrial membrane. Thus, motile IP3Rs can traffic in and out of ERMCs, and, when 'parked', mediate calcium signal propagation to the mitochondria, creating a dynamic arrangement that supports local communication.


Asunto(s)
Señalización del Calcio , Mitocondrias , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Señalización del Calcio/fisiología , Mitocondrias/metabolismo , Respiración de la Célula , Calcio/metabolismo , Estrés Oxidativo
9.
Front Neurosci ; 16: 828571, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35386592

RESUMEN

Intrauterine growth retardation (IUGR) poses a high risk for developing late-onset, non-obese type 2 diabetes (T2DM). The exact mechanism underlying this phenomenon is unknown, although the contribution of the central nervous system is recognized. The main hypothalamic nuclei involved in the homeostatic regulation express nesfatin-1, an anorexigenic neuropeptide and identified regulator of blood glucose level. Using intrauterine protein restricted rat model (PR) of IUGR, we investigated, whether IUGR alters the function of nesfatin-1. We show that PR rats develop fat preference and impaired glucose homeostasis by adulthood, while the body composition and caloric intake of normal nourished (NN) and PR rats are similar. Plasma nesfatin-1 levels are unaffected by IUGR in both neonates and adults, but pro-nesfatin-1 mRNA expression is upregulated in the hypothalamus of adult PR animals. We find that centrally injected nesfatin-1 inhibits the fasting induced neuronal activation in the hypothalamic arcuate nucleus in adult NN rats. This effect of nesfatin-1 is not seen in PR rats. The anorexigenic effect of centrally injected nesfatin-1 is also reduced in adult PR rats. Moreover, chronic central nesfatin-1 administration improves the glucose tolerance and insulin sensitivity in NN rats but not in PR animals. Birth dating of nesfatin-1 cells by bromodeoxyuridine (BrDU) reveals that formation of nesfatin-1 cells in the hypothalamus of PR rats is disturbed. Our results suggest that adult PR rats acquire hypothalamic nesfatin-1-resistance, probably due to the altered development of the hypothalamic nesfatin-1 cells. Hypothalamic nesfatin-1-resistance, in turn, may contribute to the development of non-obese type T2DM.

10.
PLoS Comput Biol ; 18(4): e1010021, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35404937

RESUMEN

Comparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature-based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous. To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity-and not inverse similarity-between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism. Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Antivirales/farmacología , Antivirales/uso terapéutico , Membrana Celular , Colesterol , Reposicionamiento de Medicamentos/métodos , Humanos
11.
STAR Protoc ; 3(1): 101119, 2022 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-35098166

RESUMEN

This protocol describes how to visualize, detect, and analyze redox signals (oxidative bursts) at the ER-mitochondrial interface. It uses drug-inducible crosslinking to target the genetically encoded glutathione redox sensor Grx1roGFP2 to organellar contact sites to measure local redox changes associated with transient depolarizations of the mitochondrial membrane potential (flickers). The strategy allows imaging of the oxidized to reduced glutathione ratio (GSSG:GSH) in subcellular regions below the diffraction limit with good temporal resolution and minimum phototoxicity. Moreover, the strategy also applies to diverse parameters including pH, H2O2, and Ca2+. For complete details on the use and execution of this profile, please refer to Booth et al. (2016) and Booth et al. (2021).


Asunto(s)
Imagen Óptica/métodos , Orgánulos/metabolismo , Transducción de Señal , Células Hep G2 , Humanos , Cinética , Oxidación-Reducción
12.
Nat Commun ; 12(1): 6064, 2021 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-34663815

RESUMEN

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAß1. We show that unlike canonical cytosolic calcineurin, CNAß1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAß1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAß1.


Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Membrana Celular/metabolismo , Lipoilación/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Calcineurina/metabolismo , Línea Celular , Citoplasma/metabolismo , Aparato de Golgi/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Unión Proteica , Isoformas de Proteínas/metabolismo , Transducción de Señal/fisiología
13.
Mol Cell ; 81(18): 3866-3876.e2, 2021 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-34352204

RESUMEN

The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IP3Rs), we show that Ca2+ channels directly sense oxidative bursts and respond with Ca2+ transients adjacent to active mitochondria. Application of specific mitochondrial stressors or apoptotic stimuli dramatically increases the frequency and amplitude of the oxidative bursts by enhancing transient permeability transition pore openings. Conversely, blocking interface Ca2+ transport via elimination of IP3Rs or mitochondrial calcium uniporter channels suppresses ER-mitochondrial Ca2+ feedback and cell death. Thus, single mitochondria initiate local retrograde signaling by miniature oxidative bursts and, upon metabolic or apoptotic stress, may also amplify signals to the rest of the cell.


Asunto(s)
Mitocondrias/metabolismo , Transporte de Proteínas/fisiología , Estallido Respiratorio/fisiología , Calcio/metabolismo , Canales de Calcio , Señalización del Calcio/fisiología , Permeabilidad de la Membrana Celular/fisiología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/fisiología , Células HEK293 , Células Hep G2 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Membranas Mitocondriales/metabolismo , Oxidación-Reducción , Estallido Respiratorio/genética , Análisis de la Célula Individual/métodos
14.
J Biol Chem ; 296: 100366, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33545176

RESUMEN

Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer-based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligand‒receptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development.


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Luciferasas/química , Luciferasas/metabolismo , Bioensayo , Membrana Celular/metabolismo , Transferencia de Energía , Células HEK293 , Humanos , Cinética , Ligandos , Unión Proteica , Transporte de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal
15.
Clin Pharmacol Ther ; 110(5): 1180-1189, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-33216976

RESUMEN

The rapid evolution of science and technology allows innovative approaches to generate new types of evidence about the effectiveness of medical product development so as to speed up patients' access to better diagnostics and treatment. Our study explored how two emerging approaches, the use of real-world evidence (RWE) and complex clinical trial (CCT) design, are currently being used by the pharmaceutical industry to support premarketing authorization of medical product development and reviewed the international landscape for regulatory acceptance of such novel approaches. Combining evidence from a literature review, company survey, and interviews with international regulators and experts, we found that 80% of Europe-based pharmaceutical companies have used RWE and 50% have used CCTs, in some capacity. Further, we present case examples of how companies are using these approaches and how international regulators are preparing for such developments. To conclude, we provide a set of recommendations for European industry and regulators to consider so that these novel approaches achieve their full potential within the EU regulatory system.


Asunto(s)
Ensayos Clínicos como Asunto/métodos , Análisis de Datos , Industria Farmacéutica/métodos , Ensayos Clínicos como Asunto/estadística & datos numéricos , Industria Farmacéutica/estadística & datos numéricos , Europa (Continente)/epidemiología , Humanos
16.
Brain Struct Funct ; 225(3): 969-984, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32200401

RESUMEN

Chronic hypernatremia activates the central osmoregulatory mechanisms and inhibits the function of the hypothalamic-pituitary-adrenal (HPA) axis. Noradrenaline (NE) release into the periventricular anteroventral third ventricle region (AV3V), the supraoptic (SON) and hypothalamic paraventricular nuclei (PVN) from efferents of the caudal ventrolateral (cVLM) and dorsomedial (cDMM) medulla has been shown to be essential for the hypernatremia-evoked responses and for the HPA response to acute restraint. Notably, the medullary NE cell groups highly coexpress prolactin-releasing peptide (PrRP) and nesfatin-1/NUCB2 (nesfatin), therefore, we assumed they contributed to the reactions to chronic hypernatremia. To investigate this, we compared two models: homozygous Brattleboro rats with hereditary diabetes insipidus (DI) and Wistar rats subjected to chronic high salt solution (HS) intake. HS rats had higher plasma osmolality than DI rats. PrRP and nesfatin mRNA levels were higher in both models, in both medullary regions compared to controls. Elevated basal tyrosine hydroxylase (TH) expression and impaired restraint-induced TH, PrRP and nesfatin expression elevations in the cVLM were, however, detected only in HS, but not in DI rats. Simultaneously, only HS rats exhibited classical signs of chronic stress and severely blunted hormonal reactions to acute restraint. Data suggest that HPA axis responsiveness to restraint depends on the type of hypernatremia, and on NE capacity in the cVLM. Additionally, NE and PrRP signalization primarily of medullary origin is increased in the SON, PVN and AV3V in HS rats. This suggests a cooperative action in the adaptation responses and designates the AV3V as a new site for PrRP's action in hypernatremia.


Asunto(s)
Adaptación Fisiológica , Hipernatremia/fisiopatología , Hipotálamo/fisiopatología , Bulbo Raquídeo/fisiopatología , Nucleobindinas/fisiología , Hormona Liberadora de Prolactina/fisiología , Animales , Masculino , Nucleobindinas/análisis , Hormona Liberadora de Prolactina/análisis , Ratas Brattleboro , Ratas Wistar , Estrés Psicológico/metabolismo , Tirosina 3-Monooxigenasa/análisis
17.
J Cell Sci ; 133(6)2020 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-32041906

RESUMEN

Oxysterol-binding protein (OSBP)-related proteins (ORPs) mediate non-vesicular lipid transfer between intracellular membranes. Phosphoinositide (PI) gradients play important roles in the ability of OSBP and some ORPs to transfer cholesterol and phosphatidylserine between the endoplasmic reticulum (ER) and other organelle membranes. Here, we show that plasma membrane (PM) association of ORP3 (also known as OSBPL3), a poorly characterized ORP family member, is triggered by protein kinase C (PKC) activation, especially when combined with Ca2+ increases, and is determined by both PI(4,5)P2 and PI4P After activation, ORP3 efficiently extracts PI4P and to a lesser extent phosphatidic acid from the PM, and slightly increases PM cholesterol levels. Full activation of ORP3 resulted in decreased PM PI4P levels and inhibited Ca2+ entry via the store-operated Ca2+ entry pathway. The C-terminal region of ORP3 that follows the strictly defined lipid transfer domain was found to be critical for the proper localization and function of the protein.


Asunto(s)
Retículo Endoplásmico , Oxidorreductasas , Fosfatos de Fosfatidilinositol , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación
18.
Proc Natl Acad Sci U S A ; 116(42): 21120-21130, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31570576

RESUMEN

Interleukin-2 (IL-2) and IL-15 play pivotal roles in T cell activation, apoptosis, and survival, and are implicated in leukemias and autoimmune diseases. Their heterotrimeric receptors share their ß- and γc-chains, but have distinct α-chains. Anti-IL-2Rα (daclizumab) therapy targeting cell surface-expressed receptor subunits to inhibit T cell proliferation has only brought limited success in adult T cell leukemia/lymphoma (ATL) and in multiple sclerosis. We asked whether IL-2R subunits could already preassemble and signal efficiently in the endoplasmic reticulum (ER) and the Golgi. A combination of daclizumab and anti-IL-2 efficiently blocked IL-2-induced proliferation of IL-2-dependent wild-type (WT) ATL cells but not cells transfected with IL-2, suggesting that in IL-2-producing cells signaling may already take place before receptors reach the cell surface. In the Golgi fraction isolated from IL-2-producing ATL cells, we detected by Western blot phosphorylated Jak1, Jak3, and a phosphotyrosine signal attributed to the γc-chain, which occurred at much lower levels in the Golgi of WT ATL cells. We expressed EGFP- and mCherry-tagged receptor chains in HeLa cells to study their assembly along the secretory pathway. Confocal microscopy, Förster resonance energy transfer, and imaging fluorescence cross-correlation spectroscopy analysis revealed partial colocalization and molecular association of IL-2 (and IL-15) receptor chains in the ER/Golgi, which became more complete in the plasma membrane, further confirming our hypothesis. Our results define a paradigm of intracellular autocrine signaling and may explain resistance to antagonistic antibody therapies targeting receptors at the cell surface.


Asunto(s)
Proliferación Celular/fisiología , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-2/metabolismo , Línea Celular Tumoral , Células HeLa , Humanos , Interleucina-15/metabolismo , Janus Quinasa 1/metabolismo , Janus Quinasa 3/metabolismo , Receptores de Interleucina-15/metabolismo , Transducción de Señal/fisiología
19.
Methods Mol Biol ; 1949: 23-34, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30790246

RESUMEN

There are several difficulties to face when investigating the role of phosphoinositides. Although they are present in most organelles, their concentration is very low, sometimes undetectable with the available methods; moreover, their level can quickly change upon several external stimuli. Here we introduce a newly improved lipid sensor tool-set based on the balanced expression of luciferase-fused phosphoinositide recognizing protein domains and a Venus protein targeted to the plasma membrane, allowing us to perform Bioluminescence Resonance Energy Transfer (BRET) measurements that reflect phosphoinositide changes in a population of transiently transfected cells. This method is highly sensitive, specific, and capable of semiquantitative characterization of plasma membrane phosphoinositide changes with high temporal resolution.


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia , Técnicas Biosensibles , Membrana Celular/metabolismo , Inositol/metabolismo , Animales , Línea Celular , Análisis de Datos , Humanos , Metabolismo de los Lípidos
20.
Mol Cell ; 72(4): 778-785.e3, 2018 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-30454562

RESUMEN

Proper control of the mitochondrial Ca2+ uniporter's pore (MCU) is required to allow Ca2+-dependent activation of oxidative metabolism and to avoid mitochondrial Ca2+ overload and cell death. The MCU's gatekeeping and cooperative activation is mediated by the Ca2+-sensing MICU1 protein, which has been proposed to form dimeric complexes anchored to the EMRE scaffold of MCU. We unexpectedly find that MICU1 suppresses inhibition of MCU by ruthenium red/Ru360, which bind to MCU's DIME motif, the selectivity filter. This led us to recognize in MICU1's sequence a putative DIME interacting domain (DID), which is required for both gatekeeping and cooperative activation of MCU and for cell survival. Thus, we propose that MICU1 has to interact with the D-ring formed by the DIME domains in MCU to control the uniporter.


Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Compuestos de Rutenio/farmacología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HEK293 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...