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1.
Cell ; 161(3): 674-690, 2015 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-25910214

RESUMEN

Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner.


Asunto(s)
Técnicas Citológicas , Proteínas , Células Cultivadas , Células Madre Embrionarias , Marcación de Gen , Humanos , ARN , Transducción Genética
2.
Cell ; 148(3): 421-33, 2012 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-22304913

RESUMEN

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKß-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.


Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Envejecimiento/metabolismo , Restricción Calórica , Transducción de Señal , Estilbenos/administración & dosificación , 3',5'-AMP Cíclico Fosfodiesterasas/química , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Tejido Adiposo Blanco/efectos de los fármacos , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Dieta , Intolerancia a la Glucosa/prevención & control , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Modelos Moleculares , Músculo Esquelético/efectos de los fármacos , NAD/metabolismo , Obesidad/prevención & control , Proteínas Quinasas/metabolismo , Resveratrol , Rolipram/administración & dosificación , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sirtuina 1/metabolismo
3.
Am J Hum Genet ; 110(1): 146-160, 2023 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-36608681

RESUMEN

Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132-requiring upregulation of neddylation to restore proteasomal function and proteasomal stress-led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature-delayed closure of the ischiopubic rami-correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.


Asunto(s)
Discapacidad Intelectual , Linfopenia , Humanos , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Transducción de Señal/genética , Discapacidad Intelectual/genética , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Linfopenia/genética
4.
Hum Mol Genet ; 32(21): 3063-3077, 2023 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-37552066

RESUMEN

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.


Asunto(s)
Discapacidad Intelectual , Megalencefalia , Trastornos del Neurodesarrollo , Animales , Humanos , Niño , Pez Cebra/genética , Pez Cebra/metabolismo , Caenorhabditis elegans/metabolismo , Trastornos del Neurodesarrollo/genética , Discapacidad Intelectual/genética , Fenotipo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Megalencefalia/genética , Discapacidades del Desarrollo/genética , Mutación Missense/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo
5.
Am J Hum Genet ; 105(2): 283-301, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31353023

RESUMEN

The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Hipotonía Muscular/patología , Mutación , Trastornos del Neurodesarrollo/patología , Saccharomyces cerevisiae/crecimiento & desarrollo , Adolescente , Edad de Inicio , Niño , Preescolar , Femenino , Células HeLa , Heterocigoto , Humanos , Masculino , Hipotonía Muscular/enzimología , Hipotonía Muscular/genética , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Fenotipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
6.
J Am Soc Nephrol ; 32(11): 2885-2899, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34607910

RESUMEN

BACKGROUND: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. METHODS: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). RESULTS: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. CONCLUSIONS: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.


Asunto(s)
Cardiomiopatía Dilatada/genética , Hipercalciuria/genética , Enfermedades Renales/genética , Proteínas de Unión al GTP Monoméricas/genética , Mutación Missense , Nefrocalcinosis/genética , Defectos Congénitos del Transporte Tubular Renal/genética , Serina-Treonina Quinasas TOR/metabolismo , Cardiomiopatía Dilatada/metabolismo , Femenino , Células HEK293 , Humanos , Hipercalciuria/metabolismo , Enfermedades Renales/metabolismo , Túbulos Renales Distales/metabolismo , Masculino , Modelos Moleculares , Natriuresis/genética , Nefrocalcinosis/metabolismo , Linaje , Conformación Proteica , Defectos Congénitos del Transporte Tubular Renal/metabolismo , Convulsiones/genética , Convulsiones/metabolismo , Transducción de Señal , Secuenciación del Exoma , Secuenciación Completa del Genoma
7.
Hum Mol Genet ; 28(1): 96-104, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30239721

RESUMEN

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.


Asunto(s)
Glutaminasa/genética , Glutaminasa/fisiología , Adolescente , Animales , Encéfalo/metabolismo , Catarata/genética , Preescolar , Discapacidades del Desarrollo/genética , Modelos Animales de Enfermedad , Femenino , Fibroblastos , Mutación con Ganancia de Función/genética , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/fisiología , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Masculino , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Pez Cebra
8.
Genet Med ; 23(11): 2202-2207, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34194004

RESUMEN

PURPOSE: Recessive cytosolic aminoacyl-tRNA synthetase (ARS) deficiencies are severe multiorgan diseases, with limited treatment options. By loading transfer RNAs (tRNAs) with their cognate amino acids, ARS are essential for protein translation. However, it remains unknown why ARS deficiencies lead to specific symptoms, especially early life and during infections. We set out to increase pathophysiological insight and improve therapeutic possibilities. METHODS: In fibroblasts from patients with isoleucyl-RS (IARS), leucyl-RS (LARS), phenylalanyl-RS-beta-subunit (FARSB), and seryl-RS (SARS) deficiencies, we investigated aminoacylation activity, thermostability, and sensitivity to ARS-specific amino acid concentrations, and developed personalized treatments. RESULTS: Aminoacylation activity was reduced in all patients, and further diminished at 38.5/40 °C (PLARS and PFARSB), consistent with infectious deteriorations. With lower cognate amino acid concentrations, patient fibroblast growth was severely affected. To prevent local and/or temporal deficiencies, we treated patients with corresponding amino acids (follow-up: 1/2-2 2/3rd years), and intensified treatment during infections. All patients showed beneficial treatment effects, most strikingly in growth (without tube feeding), head circumference, development, coping with infections, and oxygen dependency. CONCLUSION: For these four ARS deficiencies, we observed a common disease mechanism of episodic insufficient aminoacylation to meet translational demands and illustrate the power of amino acid supplementation for the expanding ARS patient group. Moreover, we provide a strategy for personalized preclinical functional evaluation.


Asunto(s)
Aminoacil-ARNt Sintetasas , Aminoácidos , Aminoacil-ARNt Sintetasas/genética , Aminoacilación , Humanos , ARN de Transferencia/metabolismo
9.
PLoS Biol ; 13(1): e1002038, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25603503

RESUMEN

The second messenger cAMP is known to augment glucose-induced insulin secretion. However, its downstream targets in pancreatic ß-cells have not been unequivocally determined. Therefore, we designed cAMP analogues by a structure-guided approach that act as Epac2-selective agonists both in vitro and in vivo. These analogues activate Epac2 about two orders of magnitude more potently than cAMP. The high potency arises from increased affinity as well as increased maximal activation. Crystallographic studies demonstrate that this is due to unique interactions. At least one of the Epac2-specific agonists, Sp-8-BnT-cAMPS (S-220), enhances glucose-induced insulin secretion in human pancreatic cells. Selective targeting of Epac2 is thus proven possible and may be an option in diabetes treatment.


Asunto(s)
AMP Cíclico/análogos & derivados , AMP Cíclico/química , Factores de Intercambio de Guanina Nucleótido/agonistas , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , AMP Cíclico/farmacología , Diseño de Fármacos , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Modelos Moleculares , Unión Proteica
10.
Proc Natl Acad Sci U S A ; 112(13): 3991-6, 2015 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-25829540

RESUMEN

Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in ß-adrenergic-dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2'-O-methyladenosine-3',5'-cyclic monophosphate (Φ-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of Φ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, Φ-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3'-,5'-cyclicmonophosphate (OMe-CPT). Φ-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. Φ-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT-induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.


Asunto(s)
Colorantes Fluorescentes/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ligandos , Animales , Calcio/metabolismo , Cardiomiopatías/metabolismo , Núcleo Celular/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/química , AMP Cíclico/metabolismo , Células HEK293 , Histona Desacetilasas/metabolismo , Humanos , Cinética , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Miocitos Cardíacos/metabolismo , Ratas , Ratas Wistar , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal , Transcripción Genética
11.
Handb Exp Pharmacol ; 238: 135-147, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27900608

RESUMEN

Epac1 and Epac2 are cyclic nucleotide-binding (CNB) domain containing proteins, which were originally identified as cAMP-regulated guanine nucleotide exchange factors (GEFs) for the small G-protein Rap. Therefore, Epac proteins founded next to protein kinase A (PKA) and cyclic nucleotide-regulated ion channels the third group of cAMP-responsive proteins in higher organisms. Epac proteins are involved in the regulation of several physiological processes. In particular Epac1 mediates the regulation of molecular processes underlying cell adhesion and mobility. In the pancreas activation of Epac2 potentiates the release of glucose-induced insulin secretion and received attention as a putative target for antidiabetic treatment. While the regulation of Epac by cAMP has been analysed in structural and biochemical detail, less is known on the interaction of Epac with non-canonical cyclic nucleotides. This chapter will discuss to what extent other cyclic purines than cAMP or cyclic pyrimidine could act as Epac agonists or antagonists. The focus will be on the biophysical analysis of the interaction between Epac and these cyclic nucleotides.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Nucleótidos Cíclicos/metabolismo , Sistemas de Mensajero Secundario , Animales , Sitios de Unión , Factores de Intercambio de Guanina Nucleótido/química , Humanos , Unión Proteica
12.
J Struct Biol ; 193(2): 106-14, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26687416

RESUMEN

CDC25 homology domain (CDC25-HD) containing Guanine Nucleotide Exchange Factors (GEFs) initiate signalling by small G-proteins of the Ras-family. Each GEF acts on a small subset of the G-proteins only, thus providing signalling selectivity. Rlf is a GEF with selectivity for the G-proteins RalA and RalB. Here the crystal structure of Rlf in complex with Ral is determined. The Rlf·Ral complex crystallised into two different crystal forms, which represent different steps of the exchange reaction. Thereby general insight in the CDC25-HD catalysed nucleotide exchange is obtained. In addition, the basis for the selectivity of the interaction is investigated. The exchange activity is monitored by the use of recombinant proteins. Selectivity determinants in the binding interface are identified and confirmed by a mutational study.


Asunto(s)
Proteínas de Drosophila/química , Factores de Transcripción/química , Proteínas de Unión al GTP ral/química , Cristalografía por Rayos X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Intercambio de Guanina Nucleótido , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismo
13.
J Biol Chem ; 288(30): 21729-41, 2013 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-23770673

RESUMEN

FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Activación Enzimática , Factores de Transcripción Forkhead , Células HEK293 , Humanos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Proteína Quinasa 8 Activada por Mitógenos/genética , Mutación , Células 3T3 NIH , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Proteínas de Unión al GTP ral/genética
14.
Biochem Biophys Res Commun ; 451(4): 497-502, 2014 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-25108158

RESUMEN

In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.


Asunto(s)
Toxinas Bacterianas/farmacología , GMP Cíclico/análogos & derivados , Nucleótidos Cíclicos/farmacología , Uridina Monofosfato/farmacología , Toxina de Adenilato Ciclasa/farmacología , Animales , Proteínas Bacterianas/farmacología , GMP Cíclico/farmacología , Glucosiltransferasas/farmacología , Ratas , Sistemas de Mensajero Secundario/fisiología , Células Tumorales Cultivadas
15.
Nature ; 455(7209): 124-7, 2008 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-18660803

RESUMEN

Epac proteins are activated by binding of the second messenger cAMP and then act as guanine nucleotide exchange factors for Rap proteins. The Epac proteins are involved in the regulation of cell adhesion and insulin secretion. Here we have determined the structure of Epac2 in complex with a cAMP analogue (Sp-cAMPS) and RAP1B by X-ray crystallography and single particle electron microscopy. The structure represents the cAMP activated state of the Epac2 protein with the RAP1B protein trapped in the course of the exchange reaction. Comparison with the inactive conformation reveals that cAMP binding causes conformational changes that allow the cyclic nucleotide binding domain to swing from a position blocking the Rap binding site towards a docking site at the Ras exchange motif domain.


Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , AMP Cíclico/análogos & derivados , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Tionucleótidos/química , Tionucleótidos/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/ultraestructura , Cristalografía por Rayos X , AMP Cíclico/química , AMP Cíclico/metabolismo , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/ultraestructura , Humanos , Ratones , Microscopía Electrónica , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/ultraestructura
16.
PLoS Genet ; 7(9): e1002289, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21980297

RESUMEN

In vertebrates, left-right (LR) axis specification is determined by a ciliated structure in the posterior region of the embryo. Fluid flow in this ciliated structure is responsible for the induction of unilateral left-sided Nodal activity in the lateral plate mesoderm, which in turn regulates organ laterality. Bmp signalling activity has been implied in repressing Nodal expression on the right side, however its mechanism of action has been controversial. In a forward genetic screen for mutations that affect LR patterning, we identified the zebrafish linkspoot (lin) mutant, characterized by cardiac laterality and mild dorsoventral patterning defects. Mapping of the lin mutation revealed an inactivating missense mutation in the Bmp receptor 1aa (bmpr1aa) gene. Embryos with a mutation in lin/bmpr1aa and a novel mutation in its paralogue, bmpr1ab, displayed a variety of dorsoventral and LR patterning defects with increasing severity corresponding with a decrease in bmpr1a dosage. In Bmpr1a-deficient embryos we observed bilateral expression of the Nodal-related gene, spaw, coupled with reduced expression of the Nodal-antagonist lefty1 in the midline. Using genetic models to induce or repress Bmp activity in combination with Nodal inhibition or activation, we found that Bmp and Nodal regulate lefty1 expression in the midline independently of each other. Furthermore, we observed that the regulation of lefty1 by Bmp signalling is required for its observed downregulation of Nodal activity in the LPM providing a novel explanation for this phenomenon. From these results we propose a two-step model in which Bmp regulates LR patterning. Prior to the onset of nodal flow and Nodal activation, Bmp is required to induce lefty1 expression in the midline. When nodal flow has been established and Nodal activity is apparent, both Nodal and Bmp independently are required for lefty1 expression to assure unilateral Nodal activation and correct LR patterning.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Proteínas Morfogenéticas Óseas/genética , Desarrollo Embrionario/genética , Factores de Determinación Derecha-Izquierda/genética , Proteína Nodal/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Animales , Tipificación del Cuerpo/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Factores de Determinación Derecha-Izquierda/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación Missense , Proteína Nodal/metabolismo , Transducción de Señal , Pez Cebra/genética , Proteínas de Pez Cebra/genética
17.
J Struct Biol ; 183(3): 312-319, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23891840

RESUMEN

Rlf is a guanine nucleotide exchange factor for the small G-proteins RalA and RalB and couples Ras- to Ral-signalling. Here the crystal structure of the catalytic module of Rlf consisting of a REM- and a CDC25-homology domain is determined. The structure is distinguished by an extended three stranded ß-sheet called the flagpole. The flagpole is a conserved element in the RalGDS family of guanine nucleotide exchange factors and stabilises the orientation of the REM-domain relative to the CDC25-homology domain. A proline-rich sequence in the flagpole is unique to Rlf and several proteins that interact with this sequence by SH3 domains are identified. Conformational pre-selection results in a gain of affinity and contributes to the establishment of SH3 domain selectivity.


Asunto(s)
Factores de Transcripción/química , Proteínas Adaptadoras del Transporte Vesicular/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Factores de Intercambio de Guanina Nucleótido , Humanos , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Miosina Tipo I/química , Fragmentos de Péptidos/química , Fosfolipasa C gamma/química , Unión Proteica , Estructura Secundaria de Proteína , Dominios Homologos src
18.
PLoS Biol ; 8(1): e1000283, 2010 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-20098723

RESUMEN

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.


Asunto(s)
Dendritas/metabolismo , Endosomas/metabolismo , Proteínas de Unión al GTP rab4/metabolismo , Animales , Transporte Biológico , Células COS , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Chlorocebus aethiops , Dendritas/ultraestructura , Escherichia coli/genética , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiología , Ratones , Plasticidad Neuronal , Proteínas Qa-SNARE/metabolismo , Ratas , Receptores de Glutamato/metabolismo , Porcinos , Proteínas de Unión al GTP rab4/análisis , Proteínas de Unión al GTP rab4/fisiología
19.
JIMD Rep ; 64(3): 217-222, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-37151363

RESUMEN

Glutaminase (GLS) hyperactivity was first described in 2019 in a patient with profound developmental delay and infantile cataract. Here, we describe a 4-year-old boy with GLS hyperactivity due to a de novo heterozygous missense variant in GLS, detected by trio whole exome sequencing. This boy also exhibits developmental delay without dysmorphic features, but does not have cataract. Additionally, he suffers from epilepsy with tonic clonic seizures. In line with the findings in the previously described patient with GLS hyperactivity, in vivo 3 T magnetic resonance spectroscopy (MRS) of the brain revealed an increased glutamate/glutamine ratio. This increased ratio was also found in urine with UPLC-MS/MS, however, inconsistently. This case indicates that the phenotypic spectrum evoked by GLS hyperactivity may include epilepsy. Clarifying this phenotypic spectrum is of importance for the prognosis and identification of these patients. The combination of phenotyping, genetic testing, and metabolic diagnostics with brain MRS and in urine is essential to identify new patients with GLS hyperactivity and to further extend the phenotypic spectrum of this disease.

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