Nuevas dianas de actuación de la proteína quinasa activada por AMP (AMPK)

RESUMEN En múltiples tejidos de mamíferos, la proteína quinasa activada por AMP (AMPK) controla el metabolismo de la glucosa y de los lípidos. Esta importante función de AMPK como sensor energético conservado evolutivamente y regulador clave del metabolismo estaría, además, apoyada por el papel de su ortólogo en el metabolismo de la glucosa del eucariota unicelular Saccharomyces cerevisiae. La proteína se activa en respuesta a un aumento en la relación de AMP respecto a ATP en el interior de la célula y, por lo tanto, actúa como un eficiente sensor del estado energético celular. Con el objetivo de mantener el balance energético celular, la activación de AMPK va a inhibir rutas anabólicas y otros procesos que consuman ATP, mientras que va a activar rutas catabólicas que generen ATP. Estos efectos se alcanzan tanto por fosforilación rápida y directa de enzimas metabólicas como a través de efectos a largo término en la expresión de genes y proteínas. Debido al papel central que AMPK tiene en el control del metabolismo energético, la identificación de nuevas proteínas que puedan interaccionar con ella puede ayudar al entendimiento de las funciones de AMPK. Mediante un escrutinio de doble híbrido en levadura de una genoteca de cDNA de páncreas humano y, utilizando AMPK α2 como cebo, se identificó TRIP6. TRIP6 es un coactivador transcripcional perteneciente a una familia de proteínas con dominios LIM que se localizan en placas de adhesión focal. TRIP6, además de interaccionar con la subunidad catalítica de AMPK cuando ambas proteínas están en el núcleo, también aparece fosforilado en su extremo amino-terminal por acción de AMPK in vitro. Y aunque la activación de AMPK no afecta a la localización subcelular de TRIP6, sí que incrementa sus propiedades transactivadoras. Últimamente se ha apuntado a TRIP6 como coactivador de genes regulados por NF-B. Consistentemente con estas observaciones, la actividad transcripcional de TRIP6 sobre promotores regulados por NF-B aumenta significativamente en condiciones de activación de AMPK. Dentro de los efectos metabólicos que produce AMPK, una mención especial merece su implicación en la regulación del metabolismo del glucógeno. Así, AMPK es capaz de interaccionar con R5, una de las subunidades reguladoras de la proteína fosfatasa de tipo 1 (PP1). PP1 es la responsable de la defosforilación de la glucógeno sintasa y de la glucógeno fosforilasa, conduciendo a la síntesis de glucógeno. También se ha descrito que R5 es capaz de interaccionar con laforina, una de las proteínas implicadas en la enfermedad de Lafora. La epilepsia mioclónica progresiva de tipo Lafora es un desorden autosómico recesivo de consecuencias fatales caracterizado por la presencia de acúmulos de glucógeno poco ramificado en distintos tipos celulares, denominados cuerpos de Lafora. Hasta el momento se han identificado dos genes que se encuentran mutados en la enfermedad de Lafora. El primero codifica para la proteína laforina y el segundo, para una E3 ubicuitina ligasa, denominada malina. R5 es una fosfoproteína in vivo cuyo grado de fosforilación se ve afectado por AMPK. Se desconoce todavía el residuo donde reside esta fosforilación, aunque hemos podido descartar la Ser35. La activación de AMPK conduce a una disminución del efecto glucogenogénico producido por la sobreexpresión de R5. Este efecto puede también producirse a través del complejo laforina-malina, ya que laforina interacciona y también puede ser fosforilada in vitro por AMPK, y la formación del complejo laforina-malina está regulada positivamente por AMPK. El complejo laforina-malina inhibe la producción de glucógeno debida a la sobreexpresión de R5, mediante la degradación de R5. Sin embargo, la expresión de una forma dominante negativa de AMPK puede prevenir la inhibición por parte del complejo laforina-malina de los efectos glucogenogénicos de R5. __________________________________________________________________________________________________AMP-activated protein kinase (AMPK) acts as a sensor of cellular energy charge. Once activated it switches on catabolic pathways and switches off many ATP-consuming processes (anabolic pathways) to preserve the energy status of the cell. This important function is supported by SNF1 complex, its ortologue in Saccharomyces cerevisiae. The identification of new proteins able to interact with AMPK may be useful in order to understand new AMPK roles. To identify new targets of AMPK action we have performed a two-hybrid screening of a human pancreas cDNA library, using AMPK α2 as a bait. As a result, we have identified TRIP6 as a novel target of AMPK. This protein belongs to the zyxin family of proteins located at the focal adhesion plaques. TRIP6 interacts with AMPK α2 when both proteins are in the nucleus. AMPK is able to phosphorylate in vitro TRIP6 at the N-terminus. Although AMPK activation does not affect subcellular localization of TRIP6, transcriptional co-activator properties of TRIP6 on NF-kB-regulated promoters were enhanced by AMPK action. AMPK is also involved in glycogen metabolism. AMPK interacts physically with R5, one of the glycogen targeting subunits of type 1 protein phosphatase (PP1). PP1 dephosphorylates glycogen synthase and glycogen phosphorylase, leading to glycogen synthesis. It has been also described that R5 interacts with laforin, one of the proteins implicated in Lafora disease. Lafora progressive myoclonus epilepsy is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. Lafora disease associates with mutations in two genes, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. R5 is also phosphorylated by AMPK and its phosphorylation status is modified by AMPK in vivo. AMPK activation decreases the glycogenic effect of R5, and this effect can be also produced through laforin-malin complex, because AMPK interacts and phosphorylates laforin in vitro, and the interaction between laforin and malin is regulated by AMPK. Thus, laforin-malin complex downregulates the glycogenic activity of R5 and the expression of a dominant negative form of AMPK impairs the effect of the laforin-malin complex on the glycogenic activity of R5

Influence of the ovine genital tract microbiota on the species artificial insemination outcome. A pilot study in commercial sheep farms

To date, there is a lack of research into the vaginal and sperm microbiome and its bearing on artificial insemination (AI) success in the ovine species. Using hypervariable regions V3–V4 of the 16S rRNA, we describe, for the first time, the combined effect of the ovine microbiome of both females (50 ewes belonging to five herds) and males (five AI rams from an AI center) on AI outcome. Differences in microbiota abundance between pregnant and non-pregnant ewes and between ewes carrying progesterone-releasing intravaginal devices (PRID) with or without antibiotic were tested at different taxonomic levels. The antibiotic treatment applied with the PRID only altered Streptobacillus genus abundance, which was significantly lower in ewes carrying PRID with antibiotic. Mageebacillus, Histophilus, Actinobacilllus and Sneathia genera were significantly less abundant in pregnant ewes. In addition, these genera were more abundant in two farms with higher AI failure. Species of these genera such as Actinobacillus seminis and Histophilus somni have been associated with reproductive disorders in the ovine species. These genera were not present in the sperm samples of AI rams, but were found in the foreskin samples of rams belonging to herd 2 (with high AI failure rate) indicating that their presence in ewes’ vagina could be due to prior transmission by natural mating with rams reared in the herd

Laforin, a dual specificity protein phosphatase involved in Lafora disease, is phosphorylated at Ser25 by AMP-activated protein kinase

Carlos Romá-Mateo et alt.Lafora progressive myoclonus epilepsy [LD (Lafora disease)] is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual-specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others showed that laforin and malin form a functional complex that regulates multiple aspects of glycogen metabolism, and that the interaction between laforin and malin is enhanced by conditions activating AMPK (AMP-activated protein kinase). In the present study, we demonstrate that laforin is a phosphoprotein, as indicated by two-dimensional electrophoresis, and we identify Ser25 as the residue involved in this modification. We also show that Ser25 is phosphorylated both in vitro and in vivo by AMPK. Lastly, we demonstrate that this residue plays a critical role for both the phosphatase activity and the ability of laforin to interact with itself and with previously established binding partners. The results of the present study suggest that phosphorylation of laforin-Ser25 by AMPK provides a mechanism to modulate the interaction between laforin and malin. Regulation of this complex is necessary to maintain normal glycogen metabolism. Importantly, Ser25 is mutated in some LD patients (S25P), and our results begin to elucidate the mechanism of disease in these patientsThis work was supported the Spanish Ministry of Education and Science [grant number SAF2008-01907 (to P.S.)]; the Generalitat Valenciana [grant number Prometeo 2009/051 (to P.S.)]; the National Institutes of Health [grant numbers R00NS061803, P20RR020171, R01NS070899 (to M.S.G.)]; and the University of Kentucky College of Medicine startup funds (to M.S.G.)Peer reviewe

Lafora disease E3-ubiquitin ligase malin is related to TRIM32 at both the phylogenetic and functional level

<p>Abstract</p> <p>Background</p> <p>Malin is an E3-ubiquitin ligase that is mutated in Lafora disease, a fatal form of progressive myoclonus epilepsy. In order to perform its function, malin forms a functional complex with laforin, a glucan phosphatase that facilitates targeting of malin to its corresponding substrates. While laforin phylogeny has been studied, there are no data on the evolutionary lineage of malin.</p> <p>Results</p> <p>After an extensive search for malin orthologs, we found that malin is present in all vertebrate species and a cephalochordate, in contrast with the broader species distribution previously reported for laforin. These data suggest that in addition to forming a functional complex, laforin and perhaps malin may also have independent functions. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32, which belongs to the tripartite-motif containing family of proteins. We present experimental evidence that both malin and TRIM32 share some substrates for ubiquitination, although they produce ubiquitin chains with different topologies. However, TRIM32-specific substrates were not reciprocally ubiquitinated by the laforin-malin complex.</p> <p>Conclusions</p> <p>We found that malin and laforin are not conserved in the same genomes. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32. The latter result suggests a common origin for malin and TRIM32 and provides insights into possible functional relationships between both proteins.</p

Laforin, the most common protein mutated in Lafora disease, regulates autophagy

Lafora disease (LD) is an autosomal recessive, progressive myoclonus epilepsy, which is characterized by the accumulation of polyglucosan inclusion bodies, called Lafora bodies, in the cytoplasm of cells in the central nervous system and in many other organs. However, it is unclear at the moment whether Lafora bodies are the cause of the disease, or whether they are secondary consequences of a primary metabolic alteration. Here we describe that the major genetic lesion that causes LD, loss-of-function of the protein laforin, impairs autophagy. This phenomenon is confirmed in cell lines from human patients, mouse embryonic fibroblasts from laforin knockout mice and in tissues from such mice. Conversely, laforin expression stimulates autophagy. Laforin regulates autophagy via the mammalian target of rapamycin kinase-dependent pathway. The changes in autophagy mediated by laforin regulate the accumulation of diverse autophagy substrates and would be predicted to impact on the Lafora body accumulation and the cell stress seen in this disease that may eventually contribute to cell death

Novel mutation in the NHLRC1 gene in a Malian family with a severe phenotype of Lafora disease

We studied a Malian family with parental consanguinity and two of eight siblings affected with late-childhood-onset progressive myoclonus epilepsy and cognitive decline, consistent with the diagnosis of Lafora disease. Genetic analysis showed a novel homozygous single-nucleotide variant in the NHLRC1 gene, c.560A>C, producing the missense change H187P. The changed amino acid is highly conserved, and the mutation impairs malin's ability to degrade laforin in vitro. Pathological evaluation showed manifestations of Lafora disease in the entire brain, with particularly severe involvement of the pallidum, thalamus, and cerebellum. Our findings document Lafora disease with severe manifestations in the West African population

AMP-Activated Kinase AMPK Is Expressed in Boar Spermatozoa and Regulates Motility

The main functions of spermatozoa required for fertilization are dependent on the energy status and metabolism. AMP-activated kinase, AMPK, acts a sensor and regulator of cell metabolism. As AMPK studies have been focused on somatic cells, our aim was to investigate the expression of AMPK protein in spermatozoa and its possible role in regulating motility. Spermatozoa from boar ejaculates were isolated and incubated under different conditions (38,5°C or 17°C, basal medium TBM or medium with Ca2+ and bicarbonate TCM, time from 1–24 hours) in presence or absence of AMPK inhibitor, compound C (CC, 30 µM). Western blotting reveals that AMPK is expressed in boar spermatozoa at relatively higher levels than in somatic cells. AMPK phosphorylation (activation) in spermatozoa is temperature-dependent, as it is undetectable at semen preservation temperature (17°C) and increases at 38,5°C in a time-dependent manner. AMPK phosphorylation is independent of the presence of Ca2+ and/or bicarbonate in the medium. We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa. Analysis of spermatozoa motility by CASA shows that CC treatment either in TBM or in TCM causes a significant reduction of any spermatozoa motility parameter in a time-dependent manner. Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients. CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation. Our results show that AMPK is expressed in spermatozoa at high levels and is phosphorylated under physiological conditions. Moreover, our study suggests that AMPK regulates a relevant function of spermatozoa, motility, which is essential for their ultimate role of fertilization

Laforin, a Dual Specificity Phosphatase Involved in Lafora Disease, Is Present Mainly as Monomeric Form with Full Phosphatase Activity

Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity

AMP-activated Protein Kinase Phosphorylates R5/PTG, the Glycogen Targeting Subunit of the R5/PTG-Protein Phosphatase 1 Holoenzyme, and Accelerates Its Down-regulation by the Laforin-Malin Complex*S⃞

R5/PTG is one of the glycogen targeting subunits of type 1 protein phosphatase, a master regulator of glycogen synthesis. R5/PTG recruits the phosphatase to the places where glycogen synthesis occurs, allowing the activation of glycogen synthase and the inactivation of glycogen phosphorylase, thus increasing glycogen synthesis and decreasing its degradation. In this report, we show that the activity of R5/PTG is regulated by AMP-activated protein kinase (AMPK). We demonstrate that AMPK interacts physically with R5/PTG and modifies its basal phosphorylation status. We have also mapped the major phosphorylation sites of R5/PTG by mass spectrometry analysis, observing that phosphorylation of Ser-8 and Ser-268 increased upon activation of AMPK. We have recently described that the activity of R5/PTG is down-regulated by the laforin-malin complex, composed of a dual specificity phosphatase (laforin) and an E3-ubiquitin ligase (malin). We now demonstrate that phosphorylation of R5/PTG at Ser-8 by AMPK accelerates its laforin/malin-dependent ubiquitination and subsequent proteasomal degradation, which results in a decrease of its glycogenic activity. Thus, our results define a novel role of AMPK in glycogen homeostasis