P-Rex1 is a novel substrate of the E3 ubiquitin ligase Malin associated with Lafora disease

Laforin and Malin are two proteins that are encoded by the genes EPM2A and EPM2B, respectively. Laforin is a glucan phosphatase and Malin is an E3-ubiquitin ligase, and these two proteins function as a complex. Mutations occurring at the level of one of the two genes lead to the accumulation of an aberrant form of glycogen meant to cluster in polyglucosans that go under the name of Lafora bodies. Individuals affected by the appearance of these polyglucosans, especially at the cerebral level, experience progressive neurodegeneration and several episodes of epilepsy leading to the manifestation of a fatal form of a rare disease called Lafora disease (LD), for which, to date, no treatment is available. Despite the different dysfunctions described for this disease, many molecular aspects still demand elucidation. An effective way to unknot some of the nodes that prevent the achievement of better knowledge of LD is to focus on the substrates that are ubiquitinated by the E3-ubiquitin ligase Malin. Some substrates have already been provided by previous studies based on protein-protein interaction techniques and have been associated with some alterations that mark the disease. In this work, we have used an unbiased alternative approach based on the activity of Malin as an E3-ubiquitin ligase. We report the discovery of novel bonafide substrates of Malin and have characterized one of them more deeply, namely PIP3-dependent Rac exchanger 1 (P-Rex1). The analysis conducted upon this substrate sets the genesis of the delineation of a molecular pathway that leads to altered glucose uptake, which could be one of the origin of the accumulation of the polyglucosans present in the disease.We want to thank Dr. Atanasio Pandiella (CIC-Salamanca), Dr. Manuel Rodríguez (Proteomics Unit. CIC-bioGUNE. Bizkaia. Spain), and Dr. Ch. Blattner (Institute of Toxicology and Genetics. Karlsruhe Institute of Technology. Karlsruhe. Germany) for plasmids. We also thank the support provided by SGIker Proteomics service (UPV/EHU - ERDF. EU). This work has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement 813599 (TRIM-NET). We also want to acknowledge the support of the grant from the National Institutes of Health P01 NS097197, which established the Lafora Epilepsy Cure Initiative (LECI), and a grant from la Fundació La Marató TV3 (202032), to PS; and a grant from the Spanish Ministry of Science and Innovation PID2020-112972RB-I00 to PS and MGG

Phenotypic characterization of a new EPM2A mutation (N163D)

2nd Biennial International Lafora Workshop. La Jolla, California, 23-24 de junio de 2016.Lafora disease (LD, OMIM 254780) is a fatal rare disorder characterized by epilepsy and neurodegeneration. In the vast majority of cases LD is related to mutations in either the EPM2A gene (encoding the glucan phosphatase laforin) or the EPM2B gene (encoding the E3-ubiquitin ligase malin). Alterations in these genes consist of deletions or missense and nonsense mutations. These alterations are spread all over the laforin and malin protein sequences and it remains to be shown whether they correlate with the severity of the disease. We have recently gained access to a primary fibroblast sample form a compound heterozygous EPM2A patient (Y112X/N163D), who shows a slow progression of the disease. As the Y112X mutation is related to regular progression of the disease and to our knowledge the laforin N 163D mutation is novel, here we have carried out the phenotypic characterization of the laforin N163D mutation. We have expressed the laforin-N 163D mutant in bacteria and purified the corresponding protein. Using OMFP as substrate we have observed no major changes in phosphatase activity. The mutant protein was also as stable as wild type when expressed either in bacteria or in mammalian cells. However, it showed a severe impairment in the interaction with regular laforin partners, as laforin itself, malin, R5/PTG and R6 (by yeast two-hybrid assays). Probably, this lack of interaction is the cause of the pathogenic profile of this novel mutation. We have recently reported that human primary fibroblasts from LD patients have an impairment of mitochondrial function with increased production of reactive oxygen species (ROS). In agreement with these observations we found that primary fibroblasts from EPM2A Y112X/N 163D patient presented higher levels of superoxide and lower levels of superoxide dismutase activity. The levels of thioredoxin1 (Trx1) were also decreased in the LD samples. All these results indicate that primary fibroblasts from EPM2A Y112X/N163D patient suffer from oxidative stress.This work was supported by grants from the Spanish Ministry of Education and Science (SAF2014-54604-C3-1-R) and from CIBERER to PS.Peer reviewe

Synergistic activation of AMPK prevents from polyglutamine-inducedtoxicity inCaenorhabditis elegans

11 páginas, 4 figuras. Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.phrs.2020.105105.Expression of abnormally long polyglutamine (polyQ) tracks is the source of a range of dominant neurodegenerative diseases, such as Huntington disease. Currently, there is no treatment for this devastating disease, although some chemicals, e.g., metformin, have been proposed as therapeutic solutions. In this work, we show that metformin, together with salicylate, can synergistically reduce the number of aggregates produced after polyQ expression in Caenorhabditis elegans. Moreover, we demonstrate that incubation polyQ-stressed worms with low doses of both chemicals restores neuronal functionality. Both substances are pleitotropic and may activate a range of different targets. However, we demonstrate in this report that the beneficial effect induced by the combination of these drugs depends entirely on the catalytic action of AMPK, since loss of function mutants of aak-2/AMPKα2 do not respond to the treatment. To further investigate the mechanism of the synergetic activity of metformin/salicylate, we used CRISPR to generate mutant alleles of the scaffolding subunit of AMPK, aakb-1/AMPKβ1. In addition, we used an RNAi strategy to silence the expression of the second AMPKβ subunit in worms, namely aakb-2/AMPKβ2. In this work, we demonstrated that both regulatory subunits of AMPK are modulators of protein homeostasis. Interestingly, only aakb-2/AMPKβ2 is required for the synergistic action of metformin/salicylate to reduce polyQ aggregation. Finally, we showed that autophagy acts downstream of metformin/salicylate-related AMPK activation to promote healthy protein homeostasis in worms.We thank the CGC, funded by the NIH Office of ResearchInfrastructure Programs (P40 OD010440), for worm strains. [...] RPVMis aMiguel Servet type IIresearcher (CPII16/00004) funded by Institutode Salud Carlos III (ISCIII, Madrid, Spain). Grants from the ISCIII wereused to perform this work (PI14/00949 and PI17/00011). All grantsfrom ISCIII are co-financed by the European Development RegionalFund”A way to achieve Europe”(ERDF). JBY holds a grant from theGeneralitat Valenciana and the European Social Fund (ACIF/2019/249). Some equipment used in this work has been funded in partnershipbetween the Generalitat Valenciana (Conselleria de Sanitat I SalutPública, Valencian Community, Spain) and European Funds (ERDF/FSE), through the call "Improvement of research infrastructures for rarediseases”CV FEDER 2014-2020. This work has been partially supportedby a grant from the Fundació Telemarató de la TV3 (Reference 559),which covered the work of MDS. The funds from the ISCIII are partiallysupported by the European Regional Development Fund. RPVM is also aMarie Curie fellow (CIG322034, EU). This work has been partiallysupported by a grant from the CIBERER (ACCI2016), a grant from theFundación Ramón Areces (CIVP19S8119) and anAyuda Miguel Gilgrantto RPVM (VII Convocatoria Ayudas a la Investigación MHER, 2019Peer reviewe

An empirical pipeline for personalized diagnosis of Lafora disease mutations

22 páginas, 6 figuras, 2 tablas. Contiene material suplementarioLafora disease (LD) is a fatal childhood dementia characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either EPM2A, encoding the glycogen phosphatase laforin, or EPM2B, encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many EPM2A variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing the functional consequences of laforin missense mutations in vitro using complementary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes that were supported by clinical cases. For example, F321C and G279C mutations have attenuated functional defects and are associated with slow progression. This pipeline enabled rapid characterization and classification of newly identified EPM2A mutations, providing clinicians and researchers genetic information to guide treatment of LD patients.This work was supported by the National Institutes of Health (P01 NS097197 to M.S.G., J.M.S. and P.S., R35 NS116824 to M.S.G., and F31 NS093892 to M.K.B), National Science Foundation (DBI2018007 and MCB1817414 to M.S.G.), and Epilepsy Foundation New Therapy Commercialization Grant to M.S.G. M.K.B. received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (No. 754510M). This project also received funding from the Spanish Ministry of Science and Innovation (SAF2017-83151-R to P.S. and RTI2018-095784b-100SAF to J.M.S).Peer reviewe

AMPKbeta subunits, more than just a scaffold in the formation of AMPK complex

11 páginas, 2 figuras.AMP-activated protein kinase (AMPK) is a sensor of energy status composed of a catalytic subunit (AMPK), a scaffolding subunit (AMPK) and a regulatory subunit involved in nucleotide binding (AMPK). Activation of AMPK results in enhancement of catabolic processes and downregulation of anabolic pathways with the aim to equilibrate the energy status of the cell. The study of the regulation of the activity of the AMPK complex has been traditionally focused on modifications of AMPK and AMPK subunits by post-translational changes (i.e. phosphorylation of the catalytic subunit) and allosteric activation by AMP. In this review we summarize recent reports that indicate that AMPK subunits are also critical players in AMPK function, since they can regulate the phosphorylation status and activity of the AMPK complex. AMPK1- and AMPK2-containing complexes differ in their capacity to be activated by specific drugs (i.e., A769622, salicylate) and also by the ability to undergo post-translational modifications. This selective behavior opens the possibility to design specific drugs that activate AMPK complexes containing specific -isoformsThis work was supported by a grant from the Spanish Ministry of Education and Science (SAF2011-27442) and a grant from Generalitat Valenciana (Prometeo 2009/051). T.R. is supported by a JAE-predoctoral fellowship from the Spanish Research Council (CSIC).Peer reviewe

Glycogenic activity of R6, a protein phosphatase 1 regulatory subunit, is modulated by the laforin-malin complex

10 páginas, 7 figuras.Protein phosphatase type 1 (PP1) plays a major role in the regulation of glycogen biosynthesis. PP1 is recruited to sites of glycogen formation by its binding to specific targeting subunits. There, it dephosphorylates different enzymes involved in glycogen homeostasis leading to an activation of glycogen biosynthesis. Regulation of these targeting subunits is crucial, as excess of them leads to an enhancement of the action of PP1, which results in glycogen accumulation. In this work we present evidence that PPP1R3D (R6), one of the PP1 glycogenic targeting subunits, interacts physically with laforin, a glucan phosphatase involved in Lafora disease, a fatal type of progressive myoclonus epilepsy. Binding of R6 to laforin allows the ubiquitination of R6 by the E3-ubiquitin ligase malin, what targets R6 for autophagic degradation. As a result of the action of the laforin-malin complex on R6, its glycogenic activity is downregulated. Since R6 is expressed in brain, our results suggest that the laforin-malin complex downregulates the glycogenic activity of R6 present in neuron cells to prevent glycogen accumulation.This work was supported by a grant from the Spanish Ministry of Education and Science (SAF2011-27442), a grant from la Fundació La Marato de TV3 (ref. 100130) and a grant from Generalitat Valenciana (Prometeo 2009/051).Peer reviewe

Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties

17 páginas, 7 figuras, 1 tabla.Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.This work has been supported by grants from the Spanish Ministry of Education and Science SAF2011-27442 and a grant from Generalitat Valenciana (PrometeoII/2014/029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Reactive Glia Inflammatory Signaling Pathways and Epilepsy

17 páginas, 6 figuras, 1 tablaNeuroinflammation and epilepsy are interconnected. Brain inflammation promotes neuronal hyper-excitability and seizures, and dysregulation in the glia immune-inflammatory function is a common factor that predisposes or contributes to the generation of seizures. At the same time, acute seizures upregulate the production of pro-inflammatory cytokines in microglia and astrocytes, triggering a downstream cascade of inflammatory mediators. Therefore, epileptic seizures and inflammatory mediators form a vicious positive feedback loop, reinforcing each other. In this work, we have reviewed the main glial signaling pathways involved in neuroinflammation, how they are affected in epileptic conditions, and the therapeutic opportunities they offer to prevent these disorders.This research was supported by grants from the Spanish Ministry of Economy and Competitiveness SAF2017-83151-R and a grant from the National Institute of Health (NIH-NINDS) P01NS097197, which established the Lafora Epilepsy Cure Initiative (LECI).Peer reviewe

Lafora disease: a ubiquitination-related pathology

This article belongs to the Special Issue Ubiquitination in Health and Disease.Lafora disease (LD, OMIM254780) is a rare and fatal form of progressive myoclonus epilepsy (PME). Among PMEs, LD is unique because of the rapid neurological deterioration of the patients and the appearance in brain and peripheral tissues of insoluble glycogen-like (polyglucosan) inclusions, named Lafora bodies (LBs). LD is caused by mutations in the EPM2A gene, encoding the dual phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Laforin and malin form a functional complex that is involved in the regulation of glycogen synthesis. Thus, in the absence of a functional complex glycogen accumulates in LBs. In addition, it has been suggested that the laforin-malin complex participates in alternative physiological pathways, such as intracellular protein degradation, oxidative stress, and the endoplasmic reticulum unfolded protein response. In this work we review the possible cellular functions of laforin and malin with a special focus on their role in the ubiquitination of specific substrates. We also discuss here the pathological consequences of defects in laforin or malin functions, as well as the therapeutic strategies that are being explored for LD.This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness SAF2017-83151-R, a grant from Fundación Ramón Areces and a grant from the National Institute of Health P01NS097197 to establish the Lafora Epilepsy Cure Initiative (LECI)

AMPK Protein Interaction Analyses by Yeast Two-Hybrid

This is a post-peer-review, pre-copyedit version of an article published in Methods in Molecular Biology. The final authenticated version is available online at: http://dx.doi.org/10.1007/978-1-4939-7598-3_9 (15 páginas, 5 figuras)Mammalian AMP-activated protein kinase (AMPK) is a Ser/Thr protein kinase that acts as a crucial energy sensor in the cell. Since AMPK plays a key role in a multitude of different pathways in the cell, major efforts have been concentrated to elucidate its signaling network, mainly by the identification of AMPK downstream targets. In this chapter we describe a yeast two-hybrid method for the direct evaluation of the interaction between an AMPK subunit and putative substrates.This study was supported by grants from the Spanish Ministry of Education and Science (SAF2014-54604-C3-1-R) and Generalitat Valenciana (PrometeoII/2014/029) to P.S.Peer reviewe