Neuronal Proteomic Analysis Of The Ubiquitinated Substrates Of The Disease-Linked E3 Ligases Parkin And Ube3a

Both Parkin and UBE3A are E3 ubiquitin ligases whose mutations result in severe brain dysfunction. Several of their substrates have been identified using cell culture models in combination with proteasome inhibitors, but not in more physiological settings. We recently developed the (bio)Ub strategy to isolate ubiquitinated proteins in flies and have now identified by mass spectrometry analysis the neuronal proteins differentially ubiquitinated by those ligases. This is an example of how flies can be used to provide biological material in order to reveal steady state substrates of disease causing genes. Collectively our results provide new leads to the possible physiological functions of the activity of those two disease causing E3 ligases. Particularly, in the case of Parkin the novelty of our data originates from the experimental setup, which is not overtly biased by acute mitochondrial depolarisation. In the case of UBE3A, it is the first time that a nonbiased screen for its neuronal substrates has been reported.The authors thank Michael Clague for insightful comments on an early version of the manuscript. Ugo Mayor, Nerea Osinalde, and Jesus M. Arizmendi are supported by the Spanish MINECO (Grant no. SAF2016-76898-P)

How to Inactivate Human Ubiquitin E3 Ligases by Mutation

E3 ubiquitin ligases are the ultimate enzymes involved in the transfer of ubiquitin to substrate proteins, a process that determines the fate of the modified protein. Numerous diseases are caused by defects in the ubiquitin-proteasome machinery, including when the activity of a given E3 ligase is hampered. Thus, inactivation of E3 ligases and the resulting effects at molecular or cellular level have been the focus of many studies during the last few years. For this purpose, site-specific mutation of key residues involved in either protein interaction, substrate recognition or ubiquitin transfer have been reported to successfully inactivate E3 ligases. Nevertheless, it is not always trivial to predict which mutation(s) will block the catalytic activity of a ligase. Here we review over 250 site-specific inactivating mutations that have been carried out in 120 human E3 ubiquitin ligases. We foresee that the information gathered here will be helpful for the design of future experimental strategies.This work was supported by Spanish MINECO (grant SAF2016-76898-P) cofinanced with FEDER funds. JR was funded with a postdoctoral fellowship from the University of the Basque Country (UPV/EHU)

The Ubiquitin Ligase Ariadne-1 Regulates Neurotransmitter Release Via Ubiquitination of NSF

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified sixteen candidates that met the established criteria: a significant change of at least two-fold increase on ubiquitination, with at least two unique peptides identified. Amongst those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pulldown approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss of function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared to controls in a Ca2+ dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous versus evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitinationThis research was funded by grants BFU2015-65685 and PGC2018-094630-B-100 from the Spanish Ministry of Economy to A. F. and grant SAF2016-76898-P from the Spanish Ministry of Economy cofinanced with FEDER funds to U. M. J. R. was supported with a postdoctoral research fellowship from the University of the Basque Country (UPV/EHU

SUMOylation in the control of cholesterol homeostasis

SUMOylation-protein modification by the small ubiquitin-related modifier (SUMO)-affects several cellular processes by modulating the activity, stability, interactions or subcellular localization of a variety of substrates. SUMO modification is involved in most cellular processes required for the maintenance of metabolic homeostasis. Cholesterol is one of the main lipids required to preserve the correct cellular function, contributing to the composition of the plasma membrane and participating in transmembrane receptor signalling. Besides these functions, cholesterol is required for the synthesis of steroid hormones, bile acids, oxysterols and vitamin D. Cholesterol levels need to be tightly regulated: in excess, it is toxic to the cell, and the disruption of its homeostasis is associated with various disorders like atherosclerosis and cardiovascular diseases. This review focuses on the role of SUMO in the regulation of proteins involved in the metabolism of cholesterol.We apologize to those whose related publications could not be cited due to space limitations. We are grateful to all members of Barrio's Lab for comments and suggestions. R.B. acknowledges grant nos. BFU2017-84653-P (MINECO/AEI/FEDER/EU), SEV-2016-0644 (Severo Ochoa Excellence Program, MINECO/AEI), 765445-EU (UbiCODE Program, EU) and SAF2017-90900-REDT (UBIRed Program, MINECO/AEI)

The role of SUMOylation during development

During the development of multicellular organisms, transcriptional regulation plays an important role in the control of cell growth, differentiation and morphogenesis. SUMOylation is a reversible post-translational process involved in transcriptional regulation through the modification of transcription factors and through chromatin remodelling (either modifying chromatin remodelers or acting as a `molecular glue' by promoting recruitment of chromatin regulators). SUMO modification results in changes in the activity, stability, interactions or localization of its substrates, which affects cellular processes such as cell cycle progression, DNA maintenance and repair or nucleocytoplasmic transport. This review focuses on the role of SUMO machinery and the modification of target proteins during embryonic development and organogenesis of animals, from invertebrates to mammals.We apologize to those whose related publication could not be cited due to space limitations. We are grateful to all members of Barrio's Lab for comments and suggestions. R.B. acknowledges grants BFU2017-84653-P (MINECO/AEI/FEDER/EU), SEV-2016-0644 (Severo Ochoa Excellence Program, MINECO/AEI), 765445-EU (UbiCODE Program, EU), SAF2017-90900-REDT (UBIRed Program, MINECO/AEI)

Solvent-Based Elimination of Organic Matter from Marine-Collected Plastics

The physical-chemical characterization of plastic litter from the marine environment requires the prior removal of the biofouling attached to their surface without causing any degradation in the polymer. The absence of a standardized protocol for digesting biofouling and organic matter of both macro and microplastic samples extracted from seawater has been the main motivation for this research work, which aims to evaluate the effectiveness of different solvents (hydrogen peroxide, ethanol, a commercial enzymatic detergent, and potassium hydroxide) for the digestion of organic matter and biofouling in different samples recovered from the Spanish Atlantic and Mediterranean coast. Moreover, the potential effect of those solvents on the physical-chemical structure of polymers, four virgin plastic reference materials (low-density polyethylene, polyamide, poly(ethylene terephthalate) and polystyrene) without any type of prior degradation has been characterized in terms of Fourier transform infrared spectroscopy (FTIR) and optical microscopy. Results indicate that the hydrogen peroxide at 15% concentration applied for one week at 40 °C is the most effective solvent for organic matter and biofouling removal, without causing any apparent damage on the structure of plastic samples analyzed

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

Identification of substrates for human deubiquitinating enzymes (DUBs): An up-to-date review and a case study for neurodevelopmental disorders

Similar to the reversal of kinase-mediated protein phosphorylation by phosphatases, deubiquitinating enzymes (DUBs) oppose the action of E3 ubiquitin ligases and reverse the ubiquitination of proteins. A total of 99 human DUBs, classified in 7 families, allow in this way for a precise control of cellular function and homeostasis. Ubiquitination regulates a myriad of cellular processes, and is altered in many pathological conditions. Thus, ubiquitination-regulating enzymes are increasingly regarded as potential candidates for therapeutic intervention. In this context, given the predicted easier pharmacological control of DUBs relative to E3 ligases, a significant effort is now being directed to better understand the processes and substrates regulated by each DUB. Classical studies have identified specific DUB substrate candidates by traditional molecular biology techniques in a case-by-case manner. Lately, single experiments can identify thousands of ubiquitinated proteins at a specific cellular context and narrow down which of those are regulated by a given DUB, thanks to the development of new strategies to isolate and enrich ubiquitinated material and to improvements in mass spectrometry detection capabilities. Here we present an overview of both types of studies, discussing the criteria that, in our view, need to be fulfilled for a protein to be considered as a high-confidence substrate of a given DUB. Applying these criteria, we have manually reviewed the relevant literature currently available in a systematic manner, and identified 650 high-confidence substrates of human DUBs. We make this information easily accessible to the research community through an updated version of the DUBase website (https://ehubio.ehu.eus/dubase/). Finally, in order to illustrate how this information can contribute to a better understanding of the physiopathological role of DUBs, we place a special emphasis on a subset of these enzymes that have been associated with neurodevelopmental disorders.This work was supported by Spanish MINECO grant SAF2016-76898-P to UM and by the University of the Basque Country (UPV/EHU), grant numbers US19/05 and COLAB19/18

Konpartimentu-espezifikoko gertuko biotinilazioa: XPO1en esportazio-kargoak identifikatzeko hurbilketa berria

A major feature of eukaryotic cells is the separation between nucleus and cytoplasm. Although physically separated, these two compartments are in permanent communication through a transport system that must be precisely regulated to maintain cell homeostasis and avoid serious diseases, such as cancer. A crucial element in this transport system is the exportin XPO1, which exports many proteins (so-called cargoes) from the nucleus to cytoplasm. XPO1 alteration has been frequently associated with cancer and XPO1 is an important therapeutic target. The cellular effect of XPO1 inhibition is expected to be mediated by changes in the subcellular distribution of its cargoes. However, while many of XPO1 cargoes have been already identified, the complete set remains uncharacterized, making XPO1 a very interesting candidate for proteomic studies. Thus, we have designed a novel proteomics strategy, based on compartment-specific proximity biotinylation using the APEX2 peroxidase, to search for XPO1 cargos. To this end, we have targeted APEX2 to cytoplasm and nucleus, isolated the biotinylated proteins by affinity purification, and identified them by mass spectrometry. The results of a proof-of concept experiment reported here show that this strategy, combined with specific XPO1 inhibition, can lead to the identification of XPO1 cargoes. This novel approach, therefore, may advance our understanding of XPO1-dependent nuclear export by facilitating the identification of novel cargoes, and may also contribute to better characterize the cellular effect of therapeutically used XPO1 inhibitors.; Nukleo eta zitoplasmaren arteko banaketa da zelula eukariotoen ezaugarririk bereizgarriena. Konpartimentuok fisikoki banaturik egon arren, elkarren arteko komunikazioa etengabea da, eta horretarako estuki erregulaturiko garraio-sistema dago zeina zelularen homeostasia mantendu eta minbizia bezalako gaixotasunak ekiditeko ezinbestekoa den. Garraio-sistema honetako pieza gakoa XPO1 esportina da zeinak kargo deritzen proteina askoren nukleotik zitoplasmaranzko esportazioa egikaritzen duen. Esportina honen eta minbiziaren arteko lotura maiz aipatu izan da, eta badira XPO1 inhibitzen duten agente terapeutikoak. XPO1en inhibizioak bere kargoen banaketa azpizelularrean aldaketak eragingo dituela espero daiteke. Alabaina, XPO1en kargo asko ezagunak badira ere, bere kargo bilduma osoa ez da zehaztu oraindik, eta honek XPO1 ikerketa proteomikoetarako kandidatu oso interesgarri bilakatzen du. Hau honela, estrategia proteomiko berri bat diseinatu dugu, zeinetan APEX2 peroxidasa erabiliz konpartimentu-espezifikoko gertuko biotinilazioa burutu dugun XPO1en kargo berriak bilatzeko. Horretarako, APEX2 zitoplasma eta nukleora ituratu dugu eta, biotinilatutako proteinak afinitate-purifikazioz arrantzatu ostean, masa-espektrometriaz identifikatu ditugu. Lan honetan azaldutako kontzeptu-froga esperimentuaren emaitzek erakusten dute estrategia hau, XPO1en inhibizio espezifikoarekin konbinatuz, kargo berriak identifikatzeko baliagarria izan daitekeela. Hurbilketa berri honek beraz, kargo gehiagoren identifikazioa erraztearekin batera, XPO1en mendeko garraioan sakondu eta terapeutikoki erabiltzen diren XPO1en inhibitzaileek zelula mailan duten eragina argitzeko balio dezake ere