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

Búsqueda y caracterización de sustratos fisiológicos de malina, la E3-ubiquitin ligasa implicada en la enfermedad de Lafora

La enfermedad de Lafora es una rara enfermedad genética que afecta principalmente a los adolescentes y pertenece al grupo de enfermedades conocidas como Epilepsias Mioclónicas Progresivas (PME). Es una forma fatal de epilepsia mioclónica progresiva y tiene una incidencia de menos de 4 casos en un millón de personas en todo el mundo. La LD es causada por la acumulación de inclusiones aberrantes similares al glucógeno conocidas como cuerpos de Lafora (LB), que están presentes en varios tejidos pero se encuentran predominantemente en el cerebro. Estos LB son insolubles y su agregación conduce a la toxicidad celular, generando varios síntomas neurológicos progresivos, que incluyen convulsiones de difícil control, mioclonías, ataxia, demencia y otros síntomas. Actualmente no existe una cura definitiva para la LD, y el tratamiento es principalmente sintomático y de apoyo, centrándose en controlar las convulsiones y manejar otros síntomas a medida que surgen. La enfermedad es causada por mutaciones en genes que codifican para dos proteínas diferentes: Laforin y Malin. Estas proteínas tienen diferentes funciones pero trabajan en forma compleja entre sí. Durante el doctorado, los estudios se centraron especialmente en Malina, conocida por ser una ubiquitina ligasa E3 que desempeña un papel importante en un proceso llamado ubiquitinación. Por lo tanto, la actividad de Malina convierte la enfermedad de Lafora en una enfermedad relacionada con el sistema de ubiquitinación. Se han identificado varios sustratos de Malina hasta ahora, incluidos los implicados en la acumulación de poliglucosanos, deterioro en los procesos de degradación a nivel del proteasoma y autofagia, alteración de la transmisión glutamatérgica y disfunción mitocondrial. Sin embargo, muchos mecanismos moleculares que conducen a estas condiciones necesitan mayor aclaración. La búsqueda de nuevos sustratos podría ayudar a identificar disfunciones de la enfermedad de Lafora no identificadas previamente y a una mayor comprensión de las alteraciones fisiopatológicas descritas anteriormente. Un análisis proteómico utilizando la estrategia bioUb identificó 88 candidatos potenciales diferencialmente ubiquitinados involucrados en el plegamiento de proteínas, la respuesta al choque térmico y la regulación de la función mitocondrial. Se eligieron dos proteínas, P-Rex1 y Hsp90α, para su posterior estudio debido a su alta tasa de ubiquitinación y número de péptido único en el análisis proteómico. En esta tesis, se reportarán evidencias para demostrar cómo el primer sustrato se relaciona con la enfermedad de Lafora. Se ha validado la ubiquitinación de P-Rex1 dependiente de Malina y hemos estudiado como esta modificación altera la actividad de P-Rex1 como factor intercambiador de nucleótidos de guanina (GEF) sobre la GTPasa Rac1 y en la toma de glucosa. El análisis realizado sobre este sustrato establece la génesis de una vía molecular que conduce a la alteración de la captación de glucosa, lo que podría ser uno de los orígenes de la acumulación de los poliglucosanos presentes en la enfermedad. Los experimentos realizados para Hsp90α la han validado como sustrato de Malina y, hemos hipotetizado cómo podría estar relacionada con la enfermedad.Lafora disease (LD) is a rare genetic disease that mainly affects adolescents and belongs to the group of diseases known as Progressive Myoclonus Epilepsies (PMEs). It is a fatal form of progressive myoclonus epilepsy and has an incidence of less than 4 cases in a million people worldwide. LD is caused by the accumulation of aberrant glycogen-like inclusions known as Lafora bodies (LBs), which are present in several tissues but are predominantly found in the brain. These LBs are insoluble and their aggregation leads to cellular toxicity, generating several progressive neurological symptoms, including difficult-to-control seizures, myoclonus, ataxia, dementia, and other symptoms. There is currently no definitive cure for LD, and treatment is mainly symptomatic and supportive, focusing on controlling seizures and managing other symptoms as they arise. The disease is caused by mutations that fall onto genes that codify for two different proteins: Laforin and Malin. These proteins have different functions but work in complex with each other. My Ph.D. studies focused especially on Malin, known to be an E3 ubiquitin ligase which plays a major role in a process called ubiquitination. Therefore, Malin’s activity makes LD a disease connected to the ubiquitin system. Several substrates of Malin have been identified to date, including those involved in the accumulation of polyglucosans, impairment in the degradation processes at the level of the proteasome and autophagy, alteration of glutamatergic transmission and mitochondrial dysfunction. However, many molecular mechanisms leading to these conditions need further elucidation. The hunt for novel substrates could help to identify previously unidentified dysfunctions of Lafora disease and to gain a better understanding of the aforementioned pathophysiological alterations. A proteomic analysis using the bioUb strategy identified 88 differentially ubiquitinated potential candidates involved in protein folding, heat shock response, and regulation of mitochondrial function. Two proteins, P-Rex1 and Hsp90α, were chosen for further study due to their high ubiquitination rate and/or unique peptide number in the proteomic analysis. In this thesis, evidence will be reported in demonstrating how the first substrate is related to LD. We have validated the Malin-dependent ubiquitination of P-Rex1 and have focused on the effect of Malin on the function of P-Rex1 as a guanine-nucleotide exchange factor (GEF) in activating Rac1 GTPase and in increasing glucose uptake. 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 origins of the accumulation of the polyglucosans present in the disease. Experiments conducted for Hsp90α have validated it as a substrate of Malin, not only when it is overexpressed but also at endogenous level and, further on, we have hypothesized how it could possibly be related to the disease

TRIM32 and Malin in Neurological and Neuromuscular Rare Diseases

Tripartite motif (TRIM) proteins are RING E3 ubiquitin ligases defined by a shared domain structure. Several of them are implicated in rare genetic diseases, and mutations in TRIM32 and TRIM-like malin are associated with Limb-Girdle Muscular Dystrophy R8 and Lafora disease, respectively. These two proteins are evolutionary related, share a common ancestor, and both display NHL repeats at their C-terminus. Here, we revmniew the function of these two related E3 ubiquitin ligases discussing their intrinsic and possible common pathophysiological pathways

Identification of novel substrates of Malin E3 ubiquitin ligase in Lafora disease

43rd Congress of the SEBBM Virtual, 19-22 de julio de 2021Lafora disease (LD) is a progressive neurological disorder characterized by epileptic seizures, myoclonus, cerebellar symptoms and psychic deterioration. There is no cure and patients are treated in a palliative way with anti-epileptic drugs, towards which, after some time, they become re sistant. At the basis of the disease, there is a malfunction of two proteins, laforin and malin, encoded respectively by two genes: EPM2A and EPM2B. The two proteins form a complex and its incorrect functionality generates an error in the metabolism of glycogen leading to the accumulation of polyglucosan inclusions in patients. The polyglucosans, have an anomalous structure that prevents its normal deg radation leading to the formation of Lafora bodies. Studies conducted on brain samples of LD mouse models show a greater accumulation of polyglucosans at the level of astro cytes compared to neurons. Considering the role of malin, known to be an E3 ubiquitin ligase, involved the ubiquit ination of specific substrates, we performed a proteomic analysis of the enriched ubiquitinated fraction of proteins, in HEK293T cells expressing either wild type or an inactive form of malin carrying the pathogenic P69A mutation (the most prevalent mutation of EPM2B gene). In comparison to cells expressing the non-functional malin-P69A, a list of more ubiquitinated putative substrates in cells expressing wild type malin was obtained.The aim of this study is to validate these candidates as substrates of malin and then focus on the consequences of ubiquitination on their physiological function. This infor mation will allow the identification of putative therapeutic targets and develop new treatments that could ameliorate the pathology present in Lafora disease.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813599)Peer reviewe

Deciphering the Polyglucosan Accumulation Present in Lafora Disease Using an Astrocytic Cellular Model

15 páginas, 7 figuras, 1 tablaLafora disease (LD) is a neurological disorder characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of insoluble forms of glycogen (polyglucosan bodies, or PGBs) in the brain. The accumulation of PGBs is causative of the pathophysiological features of LD. However, despite the efforts made by different groups, the question of why PGBs accumulate in the brain is still unanswered. We have recently demonstrated that, in vivo, astrocytes accumulate most of the PGBs present in the brain, and this could lead to astrocyte dysfunction. To develop a deeper understanding of the defects present in LD astrocytes that lead to LD pathophysiology, we obtained pure primary cultures of astrocytes from LD mice from the postnatal stage under conditions that accumulate PGBs, the hallmark of LD. These cells serve as novel in vitro models for studying PGBs accumulation and related LD dysfunctions. In this sense, the metabolomics of LD astrocytes indicate that they accumulate metabolic intermediates of the upper part of the glycolytic pathway, probably as a consequence of enhanced glucose uptake. In addition, we also demonstrate the feasibility of using the model in the identification of different compounds that may reduce the accumulation of polyglucosan inclusions.This work was supported by a grant from the Spanish Ministry of Science and Innovation PID2020-112972RB-I00, a grant from la Fundació La Marató TV3 (202032), and a grant from the National Institutes of Health P01NS097197, which established the Lafora Epilepsy Cure Initiative (LECI), to P.S.Peer reviewe

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research