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1.
Biochem J ; 478(23): 4099-4118, 2021 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-34704599

RESUMEN

Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism, probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterisation of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 on the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared with wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.


Asunto(s)
Proteínas Mitocondriales/metabolismo , Mitofagia , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/metabolismo , Tioléster Hidrolasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Fibroblastos , Humanos
2.
Int J Mol Sci ; 23(20)2022 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-36292958

RESUMEN

The quality control of mitochondria is critical for the survival of cells, and defects in the pathways required for this quality control can lead to severe disease. A key quality control mechanism in cells is mitophagy, which functions to remove damaged mitochondria under conditions of various stresses. Defective mitophagy can lead to a number of diseases including neurodegeneration. It has been proposed that an enhancement of mitophagy can improve cell survival, enhance neuronal function in neurodegeneration and extend health and lifespans. In this review, we highlight the role of deubiquitinating enzymes (DUBs) in the regulation of mitophagy. We summarise the current knowledge on DUBs that regulate mitophagy as drug targets and provide a list of small molecule inhibitors that are valuable tools for the further development of therapeutic strategies targeting the mitophagy pathway in neurodegeneration.


Asunto(s)
Mitofagia , Ubiquitina-Proteína Ligasas , Mitofagia/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Mitocondrias/metabolismo , Enzimas Desubicuitinizantes/metabolismo
3.
Eur Respir J ; 58(4)2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-33795320

RESUMEN

BACKGROUND: Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies. METHODS: We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. RESULTS: Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro. CONCLUSION: Our study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.


Asunto(s)
Trastornos de la Motilidad Ciliar , Síndrome de Kartagener , Cilios , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/tratamiento farmacológico , Trastornos de la Motilidad Ciliar/genética , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/tratamiento farmacológico , Síndrome de Kartagener/genética , Depuración Mucociliar
4.
FASEB J ; 34(6): 8139-8154, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32329133

RESUMEN

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre-clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non-human or non-neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra-nuclear inclusions in a polyQ length-dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.


Asunto(s)
Proteína Huntingtina/metabolismo , Cuerpos de Inclusión/metabolismo , Mitocondrias/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Células Cultivadas , Humanos , Enfermedad de Huntington/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Péptidos/metabolismo
5.
EMBO Rep ; 20(11): e47967, 2019 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-31566294

RESUMEN

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.


Asunto(s)
Evaluación Preclínica de Medicamentos , Distroglicanos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Distroglicanos/genética , Edición Génica , Marcación de Gen , Sitios Genéticos , Glicosilación/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Imagen Molecular , Distrofias Musculares/tratamiento farmacológico , Distrofias Musculares/etiología , Distrofias Musculares/metabolismo , Mutación , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo
6.
J Biol Chem ; 294(34): 12610-12621, 2019 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-31315929

RESUMEN

Microtubule-associated protein 1 light chain 3 α (LC3)/GABA type A receptor-associated protein (GABARAP) comprises a family of ubiquitin-like proteins involved in (macro)autophagy, an important intracellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane vesicles called autophagosomes. The only currently known cellular molecules covalently modified by LC3/GABARAP are membrane phospholipids such as phosphatidylethanolamine in the autophagosome membrane. Autophagy-related 4 cysteine peptidase (ATG4) proteases process inactive pro-LC3/GABARAP before lipidation, and the same proteases can also deconjugate LC3/GABARAP from lipids. To determine whether LC3/GABARAP has other molecular targets, here we generated a pre-processed LC3B mutant (Q116P) that is resistant to ATG4-mediated deconjugation. Upon expression in human cells and when assessed by immunoblotting under reducing and denaturing conditions, deconjugation-resistant LC3B accumulated in multiple forms and at much higher molecular weights than free LC3B. We observed a similar accumulation when pre-processed versions of all mammalian LC3/GABARAP isoforms were expressed in ATG4-deficient cell lines, suggesting that LC3/GABARAP can attach also to other larger molecules. We identified ATG3, the E2-like enzyme involved in LC3/GABARAP lipidation, as one target of conjugation with multiple copies of LC3/GABARAP. We show that LC3B-ATG3 conjugates are distinct from the LC3B-ATG3 thioester intermediate formed before lipidation, and we biochemically demonstrate that ATG4B can cleave LC3B-ATG3 conjugates. Finally, we determined ATG3 residue Lys-243 as an LC3B modification site. Overall, we provide the first cellular evidence that mammalian LC3/GABARAP post-translationally modifies proteins akin to ubiquitination ("LC3ylation"), with ATG4 proteases acting like deubiquitinating enzymes to counteract this modification ("deLC3ylation").


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Cisteína Endopeptidasas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Ubiquitinas/metabolismo , Células HeLa , Humanos , Peso Molecular , Mutación/genética , Especificidad por Sustrato
7.
PLoS Pathog ; 13(7): e1006460, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28727807

RESUMEN

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.


Asunto(s)
Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/inmunología , Digoxina/farmacología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Integración Viral/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Activación de Linfocitos/efectos de los fármacos , Latencia del Virus/efectos de los fármacos
8.
Nat Chem Biol ; 11(6): 387-97, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25978996

RESUMEN

Research in signal transduction aims to identify the functions of different signaling pathways in physiological and pathological states. Traditional techniques using biochemical, genetic or cell biological approaches have made important contributions to our understanding of cellular signaling. However, the single-gene approach does not take into account the full complexity of cell signaling. With the availability of omics techniques, great progress has been made in understanding signaling networks. Omics approaches can be classified into two categories: 'molecular profiling', including genomic, proteomic, post-translational modification and interactome profiling; and 'molecular perturbation', including genetic and functional perturbations.


Asunto(s)
Investigación Biomédica/métodos , Proteómica/métodos , Transducción de Señal , Análisis por Matrices de Proteínas , Proteoma/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiología
9.
Biochemistry ; 55(3): 608-17, 2016 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-26701387

RESUMEN

Protein kinases are essential regulators of most cellular processes and are involved in the etiology and progression of multiple diseases. The cdc2-like kinases (CLKs) have been linked to various neurodegenerative disorders, metabolic regulation, and virus infection, and the kinases have been recognized as potential drug targets. Here, we have developed a screening workflow for the identification of potent CLK2 inhibitors and identified compounds with a novel chemical scaffold structure, the benzobisthiazoles, that has not been previously reported for kinase inhibitors. We propose models for binding of these compounds to CLK family proteins and key residues in CLK2 that are important for the compound interactions and the kinase activity. We identified structural elements within the benzobisthiazole that determine CLK2 and CLK3 inhibition, thus providing a rationale for selectivity assays. In summary, our results will inform structure-based design of CLK family inhibitors based on the novel benzobisthiazole scaffold.


Asunto(s)
Benzotiazoles/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Benzotiazoles/síntesis química , Humanos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Alineación de Secuencia , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad
10.
BMC Biochem ; 15: 14, 2014 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-25007711

RESUMEN

BACKGROUND: Secreted luciferases are highly useful bioluminescent reporters for cell-based assays and drug discovery. A variety of secreted luciferases from marine organisms have been described that harbor an N-terminal signal peptide for release along the classical secretory pathway. Here, we have characterized the secretion of Gaussia luciferase in more detail. RESULTS: We describe three basic mechanisms by which GLUC can be released from cells: first, classical secretion by virtue of the N-terminal signal peptide; second, internal signal peptide-mediated secretion and third, non-conventional secretion in the absence of an N-terminal signal peptide. Non-conventional release of dNGLUC is not stress-induced, does not require autophagy and can be enhanced by growth factor stimulation. Furthermore, we have identified the golgi-associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1) as a suppressor of release of dNGLUC. CONCLUSIONS: Due to its secretion via multiple secretion pathways GLUC can find multiple applications as a research tool to study classical and non-conventional secretion. As GLUC can also be released from a reporter construct by internal signal peptide-mediated secretion it can be incorporated in a novel bicistronic secretion system.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Bioquímica/métodos , Luciferasas de Luciérnaga/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Bacterianas/genética , Secreciones Corporales , Genes/genética , Genes Reporteros/genética , Células HEK293 , Humanos , Luciferasas de Luciérnaga/genética , Señales de Clasificación de Proteína/genética
11.
J Vis Exp ; (196)2023 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-37458468

RESUMEN

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.


Asunto(s)
Cisteína , Neoplasias , Humanos , Proteínas Relacionadas con la Autofagia/metabolismo , Evaluación Preclínica de Medicamentos , Autofagia , Péptido Hidrolasas , Luciferasas
12.
SLAS Discov ; 28(2): 42-51, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36610640

RESUMEN

Induced pluripotent stem cells (iPSCs) have great potential as physiological disease models for human disorders where access to primary cells is difficult, such as neurons. In recent years, many protocols have been developed for the generation of iPSCs and the differentiation into specialised cell subtypes of interest. More recently, these models have been modified to allow large-scale phenotyping and high-content screening of small molecule compounds in iPSC-derived neuronal cells. Here, we describe the automated seeding of day 11 ventral midbrain progenitor cells into 96-well plates, administration of compounds, automated staining for immunofluorescence, the acquisition of images on a high-content screening platform and workflows for image analysis.


Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Células Cultivadas , Neuronas , Diferenciación Celular/fisiología , Procesamiento de Imagen Asistido por Computador
13.
bioRxiv ; 2023 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-37745522

RESUMEN

Beta-Propeller Protein-Associated Neurodegeneration (BPAN) is one of the commonest forms of Neurodegeneration with Brain Iron Accumulation, caused by mutations in the gene encoding the autophagy-related protein, WDR45. The mechanisms linking autophagy, iron overload and neurodegeneration in BPAN are poorly understood and, as a result, there are currently no disease-modifying treatments for this progressive disorder. We have developed a patient-derived, induced pluripotent stem cell (iPSC)-based midbrain dopaminergic neuronal cell model of BPAN (3 patient, 2 age-matched controls and 2 isogenic control lines) which shows defective autophagy and aberrant gene expression in key neurodegenerative, neurodevelopmental and collagen pathways. A high content imaging-based medium-throughput blinded drug screen using the FDA-approved Prestwick library identified 5 cardiac glycosides that both corrected disease-related defective autophagosome formation and restored BPAN-specific gene expression profiles. Our findings have clear translational potential and emphasise the utility of iPSC-based modelling in elucidating disease pathophysiology and identifying targeted therapeutics for early-onset monogenic disorders.

14.
ESC Heart Fail ; 9(1): 224-235, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34931757

RESUMEN

AIMS: Hippo signalling is an evolutionarily conserved pathway that controls organ size by regulating apoptosis, cell proliferation, and stem cell self-renewal. Recently, the pathway has been shown to exert powerful growth regulatory activity in cardiomyocytes. However, the functional role of this stress-related and cell death-related pathway in the human heart and cardiomyocytes is not known. In this study, we investigated the role of the transcriptional co-activators of Hippo signalling, YAP and TAZ, in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in response to cardiotoxic agents and investigated the effects of modulating the pathway on cardiomyocyte function and survival. METHODS AND RESULTS: RNA-sequencing analysis of human heart samples with doxorubicin-induced end-stage heart failure and healthy controls showed that YAP and ERBB2 (HER2) as upstream regulators of differentially expressed genes correlated with doxorubicin treatment. Thus, we tested the effects of doxorubicin on hiPSC-CMs in vitro. Using an automated high-content screen of 96 clinically relevant antineoplastic and cardiotherapeutic drugs, we showed that doxorubicin induced the highest activation of YAP/TAZ nuclear translocation in both hiPSC-CMs and control MCF7 breast cancer cells. The overexpression of YAP rescued doxorubicin-induced cell loss in hiPSC-CMs by inhibiting apoptosis and inducing proliferation. In contrast, silencing of YAP and TAZ by siRNAs resulted in elevated mitochondrial membrane potential loss in response to doxorubicin. hiPSC-CM calcium transients did not change in response to YAP/TAZ silencing. CONCLUSIONS: Our results suggest that Hippo signalling is involved in clinical anthracycline-induced cardiomyopathy. Modelling with hiPSC-CMs in vitro showed similar responses to doxorubicin as adult cardiomyocytes and revealed a potential cardioprotective effect of YAP in doxorubicin-induced cardiotoxicity.


Asunto(s)
Cardiomiopatías , Factores de Transcripción , Cardiomiopatías/inducido químicamente , Cardiomiopatías/metabolismo , Cardiotoxicidad/etiología , Doxorrubicina/efectos adversos , Doxorrubicina/metabolismo , Humanos , Miocitos Cardíacos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacología , Proteínas Señalizadoras YAP
15.
Cardiovasc Res ; 118(1): 282-294, 2022 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33386841

RESUMEN

AIMS: Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. METHODS AND RESULTS: Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001). CONCLUSION: We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.


Asunto(s)
Dinaminas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hidralazina/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacos , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Dinaminas/metabolismo , Femenino , Células HeLa , Humanos , Preparación de Corazón Aislado , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal
16.
Trends Cell Biol ; 31(7): 515-516, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33972154

RESUMEN

The ATG4 proteases are key regulators of autophagy. Until recently it was thought that their main function was to mediate the processing of ATG8 family members. A new study by Nguyen et al. reveals a role for ATG4s, independent of their catalytic activity, and proposes novel functions in mediating lipid transfer and mitophagy.


Asunto(s)
Proteínas Asociadas a Microtúbulos , Péptido Hidrolasas , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia/genética , Humanos
17.
Cell Rep ; 35(12): 109275, 2021 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-34161774

RESUMEN

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions.


Asunto(s)
Canales de Calcio/metabolismo , United States Food and Drug Administration , Animales , Apoptosis/efectos de los fármacos , Bencetonio/farmacología , Neoplasias de la Mama/patología , Calcio/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Clorhidrato de Duloxetina/farmacología , Metabolismo Energético/efectos de los fármacos , Femenino , Ensayos Analíticos de Alto Rendimiento , Homeostasis/efectos de los fármacos , Humanos , Hipertrofia , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Morfolinas/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Consumo de Oxígeno/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados , Estados Unidos
18.
J Thromb Haemost ; 18(12): 3296-3308, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32881285

RESUMEN

BACKGROUND: It is long established that von Willebrand factor (VWF) is central to hemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), organelles generated in a wide range of lengths (0.5-5.0 µm). WPB size responds to physiological cues and pharmacological treatment, and VWF secretion from shortened WPBs dramatically reduces platelet and plasma VWF adhesion to an endothelial surface. OBJECTIVE: We hypothesized that WPB-shortening represented a novel target for antithrombotic therapy. Our objective was to determine whether compounds exhibiting this activity do exist. METHODS: Using a microscopy approach coupled to automated image analysis, we measured the size of WPB bodies in primary human endothelial cells treated with licensed compounds for 24 hours. RESULTS AND CONCLUSIONS: A novel approach to identification of antithrombotic compounds generated a significant number of candidates with the ability to shorten WPBs. In vitro assays of two selected compounds confirm that they inhibit the pro-hemostatic activity of secreted VWF. This set of compounds acting at a very early stage of the hemostatic process could well prove to be a useful adjunct to current antithrombotic therapeutics. Further, in the current SARS-CoV-2 pandemic, with a considerable fraction of critically ill COVID-19 patients affected by hypercoagulability, these WPB size-reducing drugs might also provide welcome therapeutic leads for frontline clinicians and researchers.


Asunto(s)
Fibrinolíticos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Tamaño de los Orgánulos/efectos de los fármacos , Cuerpos de Weibel-Palade/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Hemostasis/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Cuerpos de Weibel-Palade/metabolismo , Cuerpos de Weibel-Palade/patología , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismo
20.
Cells ; 9(1)2019 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-31878323

RESUMEN

Autophagy is an evolutionary conserved stress survival pathway that has been shown to play an important role in the initiation, progression, and metastasis of multiple cancers; however, little progress has been made to date in translation of basic research to clinical application. This is partially due to an incomplete understanding of the role of autophagy in the different stages of cancer, and also to an incomplete assessment of potential drug targets in the autophagy pathway. While drug discovery efforts are on-going to target enzymes involved in the initiation phase of the autophagosome, e.g., unc51-like autophagy activating kinase (ULK)1/2, vacuolar protein sorting 34 (Vps34), and autophagy-related (ATG)7, we propose that the cysteine protease ATG4B is a bona fide drug target for the development of anti-cancer treatments. In this review, we highlight some of the recent advances in our understanding of the role of ATG4B in autophagy and its relevance to cancer, and perform a critical evaluation of ATG4B as a druggable cancer target.


Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/fisiología , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/fisiología , Neoplasias/tratamiento farmacológico , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/efectos de los fármacos , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Invasividad Neoplásica/fisiopatología , Neoplasias/metabolismo
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