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Ugo Mayor
Ikerbasque, Basque Foundation for Science, 48009 Bilbao, Spain

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Journal article
Published: 20 July 2021 in Environments
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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.

ACS Style

Amaia Mendoza; Galder Kortaberria; Florencio Marzo; Ugo Mayor; Oihane Basurko; Cristina Peña-Rodriguez. Solvent-Based Elimination of Organic Matter from Marine-Collected Plastics. Environments 2021, 8, 68 .

AMA Style

Amaia Mendoza, Galder Kortaberria, Florencio Marzo, Ugo Mayor, Oihane Basurko, Cristina Peña-Rodriguez. Solvent-Based Elimination of Organic Matter from Marine-Collected Plastics. Environments. 2021; 8 (7):68.

Chicago/Turabian Style

Amaia Mendoza; Galder Kortaberria; Florencio Marzo; Ugo Mayor; Oihane Basurko; Cristina Peña-Rodriguez. 2021. "Solvent-Based Elimination of Organic Matter from Marine-Collected Plastics." Environments 8, no. 7: 68.

Journal article
Published: 03 May 2021 in International Journal of Molecular Sciences
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The human genome contains nearly 100 deubiquitinating enzymes (DUBs) responsible for removing ubiquitin moieties from a large variety of substrates. Which DUBs are responsible for targeting which substrates remain mostly unknown. Here we implement the bioUb approach to identify DUB substrates in a systematic manner, combining gene silencing and proteomics analyses. Silencing of individual DUB enzymes is used to reduce their ubiquitin deconjugating activity, leading to an increase of the ubiquitination of their substrates, which can then be isolated and identified. We report here quantitative proteomic data of the putative substrates of 5 human DUBs. Furthermore, we have built a novel interactive database of DUB substrates to provide easy access to our data and collect DUB proteome data from other groups as a reference resource in the DUB substrates research field.

ACS Style

Juanma Ramirez; Gorka Prieto; Anne Olazabal-Herrero; Eva Borràs; Elvira Fernandez-Vigo; Unai Alduntzin; Nerea Osinalde; Javier Beaskoetxea; Benoit Lectez; Kerman Aloria; Jose Rodriguez; Alberto Paradela; Eduard Sabidó; Javier Muñoz; Fernando Corrales; Jesus Arizmendi; Ugo Mayor. A Proteomic Approach for Systematic Mapping of Substrates of Human Deubiquitinating Enzymes. International Journal of Molecular Sciences 2021, 22, 4851 .

AMA Style

Juanma Ramirez, Gorka Prieto, Anne Olazabal-Herrero, Eva Borràs, Elvira Fernandez-Vigo, Unai Alduntzin, Nerea Osinalde, Javier Beaskoetxea, Benoit Lectez, Kerman Aloria, Jose Rodriguez, Alberto Paradela, Eduard Sabidó, Javier Muñoz, Fernando Corrales, Jesus Arizmendi, Ugo Mayor. A Proteomic Approach for Systematic Mapping of Substrates of Human Deubiquitinating Enzymes. International Journal of Molecular Sciences. 2021; 22 (9):4851.

Chicago/Turabian Style

Juanma Ramirez; Gorka Prieto; Anne Olazabal-Herrero; Eva Borràs; Elvira Fernandez-Vigo; Unai Alduntzin; Nerea Osinalde; Javier Beaskoetxea; Benoit Lectez; Kerman Aloria; Jose Rodriguez; Alberto Paradela; Eduard Sabidó; Javier Muñoz; Fernando Corrales; Jesus Arizmendi; Ugo Mayor. 2021. "A Proteomic Approach for Systematic Mapping of Substrates of Human Deubiquitinating Enzymes." International Journal of Molecular Sciences 22, no. 9: 4851.

Journal article
Published: 01 January 2021 in Journal of Biological Chemistry
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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 16 candidates that met the established criteria: a significant change of at least twofold increase on ubiquitination, with at least two unique peptides identified. Among those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pull-down 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 presynaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared with 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 and 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 ubiquitination.

ACS Style

Juanma Ramírez; Miguel Morales; Nerea Osinalde; Imanol Martínez-Padrón; Ugo Mayor; Alberto Ferrús. The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF. Journal of Biological Chemistry 2021, 296, 100408 .

AMA Style

Juanma Ramírez, Miguel Morales, Nerea Osinalde, Imanol Martínez-Padrón, Ugo Mayor, Alberto Ferrús. The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF. Journal of Biological Chemistry. 2021; 296 ():100408.

Chicago/Turabian Style

Juanma Ramírez; Miguel Morales; Nerea Osinalde; Imanol Martínez-Padrón; Ugo Mayor; Alberto Ferrús. 2021. "The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF." Journal of Biological Chemistry 296, no. : 100408.

Journal article
Published: 27 November 2020 in International Journal of Molecular Sciences
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Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl4)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process “protein polyubiquitination” is enriched after CCl4-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl4-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl4-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis.

ACS Style

Maria Mercado-Gómez; Fernando Lopitz-Otsoa; Mikel Azkargorta; Marina Serrano-Maciá; Sofia Lachiondo-Ortega; Naroa Goikoetxea-Usandizaga; Rubén Rodríguez-Agudo; David Fernández-Ramos; Maider Bizkarguenaga; Virginia Gutiérrez-De Juan; Benoît Lectez; Kerman Aloria; Jesus M. Arizmendi; Jorge Simon; Cristina Alonso; Juan J. Lozano; Matias A. Avila; Jesus M. Banales; Jose J. G. Marin; Naiara Beraza; José M. Mato; Félix Elortza; Rosa Barrio; James D. Sutherland; Ugo Mayor; María L. Martínez-Chantar; Teresa C. Delgado. Multi-Omics Integration Highlights the Role of Ubiquitination in CCl4-Induced Liver Fibrosis. International Journal of Molecular Sciences 2020, 21, 9043 .

AMA Style

Maria Mercado-Gómez, Fernando Lopitz-Otsoa, Mikel Azkargorta, Marina Serrano-Maciá, Sofia Lachiondo-Ortega, Naroa Goikoetxea-Usandizaga, Rubén Rodríguez-Agudo, David Fernández-Ramos, Maider Bizkarguenaga, Virginia Gutiérrez-De Juan, Benoît Lectez, Kerman Aloria, Jesus M. Arizmendi, Jorge Simon, Cristina Alonso, Juan J. Lozano, Matias A. Avila, Jesus M. Banales, Jose J. G. Marin, Naiara Beraza, José M. Mato, Félix Elortza, Rosa Barrio, James D. Sutherland, Ugo Mayor, María L. Martínez-Chantar, Teresa C. Delgado. Multi-Omics Integration Highlights the Role of Ubiquitination in CCl4-Induced Liver Fibrosis. International Journal of Molecular Sciences. 2020; 21 (23):9043.

Chicago/Turabian Style

Maria Mercado-Gómez; Fernando Lopitz-Otsoa; Mikel Azkargorta; Marina Serrano-Maciá; Sofia Lachiondo-Ortega; Naroa Goikoetxea-Usandizaga; Rubén Rodríguez-Agudo; David Fernández-Ramos; Maider Bizkarguenaga; Virginia Gutiérrez-De Juan; Benoît Lectez; Kerman Aloria; Jesus M. Arizmendi; Jorge Simon; Cristina Alonso; Juan J. Lozano; Matias A. Avila; Jesus M. Banales; Jose J. G. Marin; Naiara Beraza; José M. Mato; Félix Elortza; Rosa Barrio; James D. Sutherland; Ugo Mayor; María L. Martínez-Chantar; Teresa C. Delgado. 2020. "Multi-Omics Integration Highlights the Role of Ubiquitination in CCl4-Induced Liver Fibrosis." International Journal of Molecular Sciences 21, no. 23: 9043.

Other
Published: 01 May 2020
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The SARS-CoV-2 pandemic has evolved far more aggressively in countries lacking a robust testing strategy to identify infected individuals. Given the global demand for fast and reliable diagnosis to determine the carrier individuals, a stock-out scenario for a number of essential reagents/kits used along the diagnostic process has been foreseen by many organizations. Having identified the RNA extraction step as one of the key bottlenecks, we tested several alternatives that avoid the use of commercial kits for this step. The analysis showed that 2-propanol precipitation of the viral RNA, followed by one-step RT-qPCR results in a sensitivity and specificity comparable to that provided currently by automatized systems such as the COBAS 6800 system. Therefore, this simple protocol allows SARS-CoV-2 testing independently of commercial kit providers in a time and cost-effective manner. It can be readily implemented in research and/or diagnostic laboratories worldwide, provided that patient confidentiality and researcher safety are ensured. Scaling up the testing capabilities of hospitals and research facilities will identify larger numbers of infected individuals to paint a clear picture of the COVID-19 prevalence, a pre-requisite for informed policy decision making.

ACS Style

Xabier Guruceaga; Amanda Sierra; Daniel Marino; Izortze Santin; Jon Ander Nieto-Garai; Jose Ramon Bilbao; Maier Lorizate; Patricia Aspichueta; coBIG (COVID19 Basque Inter-institutional Group); Ugo Mayor; COVID-19 Basque Inter-institutional Group (coBIG); Adhara Gaminde-Blasco; Adrian Bozal-Leorri; Agustín Marín-Peña; Ainara Castellanos-Rubio; Ainhoa Iglesias-Ara; Aitor Rementeria; Ana Bernal-Chico; Andoni Ramirez-Garcia; Ane Olazagoitia-Garmendia; Asier Benito-Vicente; César Martín; Eider Bilbao Castellanos; Eneritz Rueda-Alaña; Fabio Cavaliere; Guiomar Perez De Nanclares; Igor Aurrekoetxea; Iraia Garcia-Santisteban; Irantzu Bernales; Itziar Gonzalez-Moro; Jan Tønnesen; Jimena Baleriola; Jon Vallejo-Rodríguez; Leire Aparicio-Fernandez; Leire Martin-Souto; Luis Manuel Mendoza; Maialen Areitio; Maialen Sebastian-Delacruz; Maren Ortiz-Zarragoitia; Mari Paz Serrano-Regal; Miren Basaras; Nora Fernandez-Jimenez; Olatz Pampliega; Santos Alonso Alegre; Susi Marcos; Teresa Fuertes-Mendizabal; Unai Galicia-Garcia; Uxue Perez-Cuesta; Verónica Torrano; Virginia Sierra-Torre; Xabier Buqué; Coordinators And Participants:. Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples. 2020, 1 .

AMA Style

Xabier Guruceaga, Amanda Sierra, Daniel Marino, Izortze Santin, Jon Ander Nieto-Garai, Jose Ramon Bilbao, Maier Lorizate, Patricia Aspichueta, coBIG (COVID19 Basque Inter-institutional Group), Ugo Mayor, COVID-19 Basque Inter-institutional Group (coBIG), Adhara Gaminde-Blasco, Adrian Bozal-Leorri, Agustín Marín-Peña, Ainara Castellanos-Rubio, Ainhoa Iglesias-Ara, Aitor Rementeria, Ana Bernal-Chico, Andoni Ramirez-Garcia, Ane Olazagoitia-Garmendia, Asier Benito-Vicente, César Martín, Eider Bilbao Castellanos, Eneritz Rueda-Alaña, Fabio Cavaliere, Guiomar Perez De Nanclares, Igor Aurrekoetxea, Iraia Garcia-Santisteban, Irantzu Bernales, Itziar Gonzalez-Moro, Jan Tønnesen, Jimena Baleriola, Jon Vallejo-Rodríguez, Leire Aparicio-Fernandez, Leire Martin-Souto, Luis Manuel Mendoza, Maialen Areitio, Maialen Sebastian-Delacruz, Maren Ortiz-Zarragoitia, Mari Paz Serrano-Regal, Miren Basaras, Nora Fernandez-Jimenez, Olatz Pampliega, Santos Alonso Alegre, Susi Marcos, Teresa Fuertes-Mendizabal, Unai Galicia-Garcia, Uxue Perez-Cuesta, Verónica Torrano, Virginia Sierra-Torre, Xabier Buqué, Coordinators And Participants:. Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples. . 2020; ():1.

Chicago/Turabian Style

Xabier Guruceaga; Amanda Sierra; Daniel Marino; Izortze Santin; Jon Ander Nieto-Garai; Jose Ramon Bilbao; Maier Lorizate; Patricia Aspichueta; coBIG (COVID19 Basque Inter-institutional Group); Ugo Mayor; COVID-19 Basque Inter-institutional Group (coBIG); Adhara Gaminde-Blasco; Adrian Bozal-Leorri; Agustín Marín-Peña; Ainara Castellanos-Rubio; Ainhoa Iglesias-Ara; Aitor Rementeria; Ana Bernal-Chico; Andoni Ramirez-Garcia; Ane Olazagoitia-Garmendia; Asier Benito-Vicente; César Martín; Eider Bilbao Castellanos; Eneritz Rueda-Alaña; Fabio Cavaliere; Guiomar Perez De Nanclares; Igor Aurrekoetxea; Iraia Garcia-Santisteban; Irantzu Bernales; Itziar Gonzalez-Moro; Jan Tønnesen; Jimena Baleriola; Jon Vallejo-Rodríguez; Leire Aparicio-Fernandez; Leire Martin-Souto; Luis Manuel Mendoza; Maialen Areitio; Maialen Sebastian-Delacruz; Maren Ortiz-Zarragoitia; Mari Paz Serrano-Regal; Miren Basaras; Nora Fernandez-Jimenez; Olatz Pampliega; Santos Alonso Alegre; Susi Marcos; Teresa Fuertes-Mendizabal; Unai Galicia-Garcia; Uxue Perez-Cuesta; Verónica Torrano; Virginia Sierra-Torre; Xabier Buqué; Coordinators And Participants:. 2020. "Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples." , no. : 1.

Preprint content
Published: 20 February 2020
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Hypoxia Inducible Factor (HIF) is the master transcriptional regulator that orchestrates cellular adaptation to low oxygen. HIF is tightly regulated via the stability of its α-subunit, which is subjected to oxygen-dependent proline hydroxylation by Prolyl-Hydroxylase Domain containing proteins (PHDs/EGLNs), and ultimately targeted for proteasomal degradation through poly-ubiquitination by von-Hippel-Lindau protein (pVHL). However, sustained HIF-α signalling is found in many tumours independently of oxygen availability pointing towards the relevance of non-canonical HIF-α regulators. In this study, we establish the Ubiquitin Specific Protease 29 (USP29) as direct post-translational activator of HIF-α in a variety of cancer cell lines. USP29 binds to HIF-α, decreases poly-ubiquitination and thus protects HIF-α from proteasomal degradation. Deubiquitinating activity of USP29 is essential to stabilise not only HIF-1α but also HIF-2α, via their C-termini in an oxygen/PHD/pVHL-independent manner. Furthermore, in prostate cancer samples the expression of USP29 correlates with the HIF-target gene CA9 (carbonic anhydrase 9) as well as disease progression and severity.

ACS Style

Amelie S Schober; Ines Martin-Barros; Teresa Martin-Mateos; Encarnacion Perez-Andres; Onintza Carlevaris; Sara Pozo; Ana R Cortazar; Ana M Aransay; Arkaitz Carracedo; Ugo Mayor; Violaine See; Edurne Berra. USP29 is a novel non-canonical Hypoxia Inducible Factor-α activator. 2020, 1 .

AMA Style

Amelie S Schober, Ines Martin-Barros, Teresa Martin-Mateos, Encarnacion Perez-Andres, Onintza Carlevaris, Sara Pozo, Ana R Cortazar, Ana M Aransay, Arkaitz Carracedo, Ugo Mayor, Violaine See, Edurne Berra. USP29 is a novel non-canonical Hypoxia Inducible Factor-α activator. . 2020; ():1.

Chicago/Turabian Style

Amelie S Schober; Ines Martin-Barros; Teresa Martin-Mateos; Encarnacion Perez-Andres; Onintza Carlevaris; Sara Pozo; Ana R Cortazar; Ana M Aransay; Arkaitz Carracedo; Ugo Mayor; Violaine See; Edurne Berra. 2020. "USP29 is a novel non-canonical Hypoxia Inducible Factor-α activator." , no. : 1.

Review article
Published: 04 February 2020 in Frontiers in Cell and Developmental Biology
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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.

ACS Style

Cristina Garcia-Barcena; Nerea Osinalde; Juanma Ramirez; Ugo Mayor. How to Inactivate Human Ubiquitin E3 Ligases by Mutation. Frontiers in Cell and Developmental Biology 2020, 8, 1 .

AMA Style

Cristina Garcia-Barcena, Nerea Osinalde, Juanma Ramirez, Ugo Mayor. How to Inactivate Human Ubiquitin E3 Ligases by Mutation. Frontiers in Cell and Developmental Biology. 2020; 8 ():1.

Chicago/Turabian Style

Cristina Garcia-Barcena; Nerea Osinalde; Juanma Ramirez; Ugo Mayor. 2020. "How to Inactivate Human Ubiquitin E3 Ligases by Mutation." Frontiers in Cell and Developmental Biology 8, no. : 1.

Preprint content
Published: 24 January 2020
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Ariadne-1 (Ari-1) is an essential E3 ubiquitin-ligase whose neuronal substrates are yet to be identified. We have used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics to identify putative Ari-1 substrates in Drosophila heads. Sixteen candidates met the established criteria. Amongst those, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF). Using an in vivo GFP pulldown approach, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons. The interaction results in the monoubiquitination of Comt/NSF. We also report that Ari-1 loss of function mutants display a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants is enhanced 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. The distinct Ari-1 mutant phenotypes in spontaneous versus evoked release indicate that NSF activity discriminates the two corresponding protein ensembles that mediate each mode of release. Our results, thus, provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.

ACS Style

Juanmanuel Ramirez; Miguel Morales; Nerea Osinalde; Imanol Martinez-Padron; Ugo Mayor; Alberto Ferrus. The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release. 2020, 1 .

AMA Style

Juanmanuel Ramirez, Miguel Morales, Nerea Osinalde, Imanol Martinez-Padron, Ugo Mayor, Alberto Ferrus. The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release. . 2020; ():1.

Chicago/Turabian Style

Juanmanuel Ramirez; Miguel Morales; Nerea Osinalde; Imanol Martinez-Padron; Ugo Mayor; Alberto Ferrus. 2020. "The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release." , no. : 1.

Protocol
Published: 25 September 2019 in Methods in Molecular Biology
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Regulation by ubiquitin (Ub) and ubiquitin-like (UbL) modifiers can confer their substrate proteins a myriad of assignments, such as inducing protein-protein interactions, the internalization of membrane proteins, or their degradation via the proteasome. The underlying code regulating those diverse endpoints appears to be based on the topology of the ubiquitin chains formed.Experimental characterization of the specific regulation mediated by Ub and UbLs is not trivial. The substoichiometric levels of Ub- and UbL-modified proteins greatly limit their analytical detection in a background of more abundant proteins. Therefore, modified proteins or peptides must be enriched prior to any downstream detection analysis. For that purpose, we recently developed a GFP-tag based isolation strategy. Here we illustrate the usefulness of combining GFP-tag isolation strategy with mass spectrometry (MS) to identify Ub- and UbL-modified residues within the GFP-tagged protein, as well as to uncover the types of Ub and UbL chains formed.

ACS Style

Nagore Elu; Benoit Lectez; Juanma Ramirez; Nerea Osinalde; Ugo Mayor. Mass Spectrometry-Based Characterization of Ub- and UbL-Modified Proteins. Methods in Molecular Biology 2019, 2051, 265 -276.

AMA Style

Nagore Elu, Benoit Lectez, Juanma Ramirez, Nerea Osinalde, Ugo Mayor. Mass Spectrometry-Based Characterization of Ub- and UbL-Modified Proteins. Methods in Molecular Biology. 2019; 2051 ():265-276.

Chicago/Turabian Style

Nagore Elu; Benoit Lectez; Juanma Ramirez; Nerea Osinalde; Ugo Mayor. 2019. "Mass Spectrometry-Based Characterization of Ub- and UbL-Modified Proteins." Methods in Molecular Biology 2051, no. : 265-276.

Original research article
Published: 03 May 2019 in Frontiers in Physiology
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The ubiquitin E3 ligase UBE3A has been widely reported to interact with the proteasome, but it is still unclear how this enzyme regulates by ubiquitination the different proteasomal subunits. The proteasome receptor DDI1 has been identified both in Drosophila photoreceptor neurons and in human neuroblastoma cells in culture as a direct substrate of UBE3A. Here, we further characterize this regulation, by identifying the UBE3A-dependent ubiquitination sites and ubiquitin chains formed on DDI1. Additionally, we found one deubiquitinating enzyme that is capable of reversing the action of UBE3A on DDI1. The complete characterization of the ubiquitination pathway of an UBE3A substrate is important due to the role of this E3 ligase in rare neurological disorders as Angelman syndrome.

ACS Style

Nagore Elu; Nerea Osinalde; Javier Beaskoetxea; Juanma Ramirez; Benoit Lectez; Kerman Aloria; Jose Antonio Rodriguez; Jesus M. Arizmendi; Ugo Mayor. Detailed Dissection of UBE3A-Mediated DDI1 Ubiquitination. Frontiers in Physiology 2019, 10, 1 .

AMA Style

Nagore Elu, Nerea Osinalde, Javier Beaskoetxea, Juanma Ramirez, Benoit Lectez, Kerman Aloria, Jose Antonio Rodriguez, Jesus M. Arizmendi, Ugo Mayor. Detailed Dissection of UBE3A-Mediated DDI1 Ubiquitination. Frontiers in Physiology. 2019; 10 ():1.

Chicago/Turabian Style

Nagore Elu; Nerea Osinalde; Javier Beaskoetxea; Juanma Ramirez; Benoit Lectez; Kerman Aloria; Jose Antonio Rodriguez; Jesus M. Arizmendi; Ugo Mayor. 2019. "Detailed Dissection of UBE3A-Mediated DDI1 Ubiquitination." Frontiers in Physiology 10, no. : 1.

Review article
Published: 03 November 2018 in Seminars in Cell & Developmental Biology
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Rare diseases are classified as such when their prevalence is 1:2000 or lower, but even if each of them is so infrequent, altogether more than 300 million people in the world suffer one of the ∼7000 diseases considered as rare. Over 1200 of these disorders are known to affect the brain or other parts of our nervous system, and their symptoms can affect cognition, motor function and/or social interaction of the patients; we refer collectively to them as rare neurological disorders or RNDs. We have focused this review on RNDs known to have compromised protein homeostasis pathways. Proteostasis can be regulated and/or altered by a chain of cellular mechanisms, from protein synthesis and folding, to aggregation and degradation. Overall, we provide a list comprised of above 215 genes responsible for causing more than 170 distinct RNDs, deepening on some representative diseases, including as well a clinical view of how those diseases are diagnosed and dealt with. Additionally, we review existing methodologies for diagnosis and treatment, discussing the potential of specific deubiquitinating enzyme inhibition as a future therapeutic avenue for RNDs.

ACS Style

Nerea Osinalde; Anna Duarri; Juanma Ramirez; Rosa Barrio; Guiomar Perez De Nanclares; Ugo Mayor. Impaired proteostasis in rare neurological diseases. Seminars in Cell & Developmental Biology 2018, 93, 164 -177.

AMA Style

Nerea Osinalde, Anna Duarri, Juanma Ramirez, Rosa Barrio, Guiomar Perez De Nanclares, Ugo Mayor. Impaired proteostasis in rare neurological diseases. Seminars in Cell & Developmental Biology. 2018; 93 ():164-177.

Chicago/Turabian Style

Nerea Osinalde; Anna Duarri; Juanma Ramirez; Rosa Barrio; Guiomar Perez De Nanclares; Ugo Mayor. 2018. "Impaired proteostasis in rare neurological diseases." Seminars in Cell & Developmental Biology 93, no. : 164-177.

Journal article
Published: 21 March 2018 in Human Molecular Genetics
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Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research.

ACS Style

Juanma Ramirez; Benoit Lectez; Nerea Osinalde; Monika Sivá; Nagore Elu; Kerman Aloria; Michaela Procházková; Coralia Pérez; José Martínez-Hernández; Rosa Barrio; Klára Grantz Šašková; Jesus M Arizmendi; Ugo Mayor. Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome. Human Molecular Genetics 2018, 27, 1955 -1971.

AMA Style

Juanma Ramirez, Benoit Lectez, Nerea Osinalde, Monika Sivá, Nagore Elu, Kerman Aloria, Michaela Procházková, Coralia Pérez, José Martínez-Hernández, Rosa Barrio, Klára Grantz Šašková, Jesus M Arizmendi, Ugo Mayor. Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome. Human Molecular Genetics. 2018; 27 (11):1955-1971.

Chicago/Turabian Style

Juanma Ramirez; Benoit Lectez; Nerea Osinalde; Monika Sivá; Nagore Elu; Kerman Aloria; Michaela Procházková; Coralia Pérez; José Martínez-Hernández; Rosa Barrio; Klára Grantz Šašková; Jesus M Arizmendi; Ugo Mayor. 2018. "Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome." Human Molecular Genetics 27, no. 11: 1955-1971.

Review article
Published: 06 March 2018 in BioMed Research International
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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 theUbbiostrategy 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.

ACS Style

Aitor Martinez; Juanma Ramirez; Nerea Osinalde; Jesus M. Arizmendi; Ugo Mayor. Neuronal Proteomic Analysis of the Ubiquitinated Substrates of the Disease-Linked E3 Ligases Parkin and Ube3a. BioMed Research International 2018, 2018, 1 -14.

AMA Style

Aitor Martinez, Juanma Ramirez, Nerea Osinalde, Jesus M. Arizmendi, Ugo Mayor. Neuronal Proteomic Analysis of the Ubiquitinated Substrates of the Disease-Linked E3 Ligases Parkin and Ube3a. BioMed Research International. 2018; 2018 ():1-14.

Chicago/Turabian Style

Aitor Martinez; Juanma Ramirez; Nerea Osinalde; Jesus M. Arizmendi; Ugo Mayor. 2018. "Neuronal Proteomic Analysis of the Ubiquitinated Substrates of the Disease-Linked E3 Ligases Parkin and Ube3a." BioMed Research International 2018, no. : 1-14.

Journal article
Published: 02 March 2018 in Journal of Cell Biology
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The Parkinson’s disease factors PINK1 and parkin are strongly implicated in stress-induced mitophagy in vitro, but little is known about their impact on basal mitophagy in vivo. We generated transgenic Drosophila melanogaster expressing fluorescent mitophagy reporters to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. We find that mitophagy is readily detectable and abundant in many tissues, including Parkinson’s disease–relevant dopaminergic neurons. However, we did not detect mitolysosomes in flight muscle. Surprisingly, in Pink1 or parkin null flies, we did not observe any substantial impact on basal mitophagy. Because these flies exhibit locomotor defects and dopaminergic neuron loss, our findings raise questions about current assumptions of the pathogenic mechanism associated with the PINK1/parkin pathway. Our findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure.

ACS Style

Juliette J. Lee; Alvaro Sanchez-Martinez; Aitor Martinez Zarate; Cristiane Benincá; Ugo Mayor; Michael J. Clague; Alexander J. Whitworth. Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. Journal of Cell Biology 2018, 217, 1613 -1622.

AMA Style

Juliette J. Lee, Alvaro Sanchez-Martinez, Aitor Martinez Zarate, Cristiane Benincá, Ugo Mayor, Michael J. Clague, Alexander J. Whitworth. Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. Journal of Cell Biology. 2018; 217 (5):1613-1622.

Chicago/Turabian Style

Juliette J. Lee; Alvaro Sanchez-Martinez; Aitor Martinez Zarate; Cristiane Benincá; Ugo Mayor; Michael J. Clague; Alexander J. Whitworth. 2018. "Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin." Journal of Cell Biology 217, no. 5: 1613-1622.

Preprint
Published: 12 January 2018
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Parkinson’s disease factors, PINK1 and parkin, are strongly implicated in stress-induced mitophagy in vitro, but little is known about their impact on basal mitophagy in vivo. We generated transgenic Drosophila expressing fluorescent mitophagy reporters to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. We find that mitophagy is readily detectable and abundant in many tissues including Parkinson’s disease relevant dopaminergic neurons. However, we did not detect mitolysosomes in flight muscle. Surprisingly, in Pink1 or parkin null flies we did not observe any substantial impact on basal mitophagy. As these flies exhibit locomotor defects and dopaminergic neuron loss, our findings raise questions about current assumptions of the pathogenic mechanism associated with the PINK1/Parkin pathway. Our findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure.SummaryPINK1/parkin are key mediators of stress-induced mitophagy in vitro but their impact on basal mitophagy in vivo is unclear. Novel Drosophila reporters lines reveal abundant mitophagy in many tissues including dopaminergic neurons but is unaffected by loss of PINK1/parkin.

ACS Style

Juliette J. Lee; Alvaro Sanchez-Martinez; Aitor Martinez Zarate; Cristiane Benincá; Ugo Mayor; Michael J. Clague; Alexander J. Whitworth. Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. 2018, 235077 .

AMA Style

Juliette J. Lee, Alvaro Sanchez-Martinez, Aitor Martinez Zarate, Cristiane Benincá, Ugo Mayor, Michael J. Clague, Alexander J. Whitworth. Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. . 2018; ():235077.

Chicago/Turabian Style

Juliette J. Lee; Alvaro Sanchez-Martinez; Aitor Martinez Zarate; Cristiane Benincá; Ugo Mayor; Michael J. Clague; Alexander J. Whitworth. 2018. "Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin." , no. : 235077.

Protocol
Published: 28 June 2017 in Animal Models of Drug Addiction
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Protein ubiquitination is essential for the development of neurons and their proper functioning. Indeed, its failure is associated with a number of neurological disorders. The identification of the proteins that are ubiquitinated in vivo in neurons can greatly contribute to our understanding of the roles that this modification plays in the brain. However, the low stoichiometry at which ubiquitin-modified proteins are found within the cells makes the study of this modification quite challenging. Here we describe two methodologies that have proven to be suitable approaches for the in vivo analysis of neuronal ubiquitinated proteins. The first approach is based on the in vivo biotinylation of ubiquitin and allows the isolation and enrichment of hundreds of ubiquitin conjugates. The second approach is designed to selectively isolate particular proteins in order to characterize their ubiquitinated fraction.

ACS Style

Juanma Ramirez; Nagore Elu; Aitor Martinez; Benoit Lectez; Ugo Mayor. In Vivo Strategies to Isolate and Characterize the Neuronal Ubiquitinated Proteome. Animal Models of Drug Addiction 2017, 179 -189.

AMA Style

Juanma Ramirez, Nagore Elu, Aitor Martinez, Benoit Lectez, Ugo Mayor. In Vivo Strategies to Isolate and Characterize the Neuronal Ubiquitinated Proteome. Animal Models of Drug Addiction. 2017; ():179-189.

Chicago/Turabian Style

Juanma Ramirez; Nagore Elu; Aitor Martinez; Benoit Lectez; Ugo Mayor. 2017. "In Vivo Strategies to Isolate and Characterize the Neuronal Ubiquitinated Proteome." Animal Models of Drug Addiction , no. : 179-189.

Editorial
Published: 31 May 2017 in Oncotarget
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ACS Style

Aitor Martinez; Ugo Mayor; Michael J. Clague. Multi-story Parkin. Oncotarget 2017, 8, 50327 -50328.

AMA Style

Aitor Martinez, Ugo Mayor, Michael J. Clague. Multi-story Parkin. Oncotarget. 2017; 8 (31):50327-50328.

Chicago/Turabian Style

Aitor Martinez; Ugo Mayor; Michael J. Clague. 2017. "Multi-story Parkin." Oncotarget 8, no. 31: 50327-50328.

Journal article
Published: 11 April 2017 in Molecular Neurodegeneration
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Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson’s Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings. Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells. We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells. Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation.

ACS Style

Aitor Martinez; Benoit Lectez; Juanma Ramirez; Oliver Popp; James D. Sutherland; Sylvie Urbé; Gunnar Dittmar; Michael J. Clague; Ugo Mayor. Quantitative proteomic analysis of Parkin substrates in Drosophila neurons. Molecular Neurodegeneration 2017, 12, 1 -19.

AMA Style

Aitor Martinez, Benoit Lectez, Juanma Ramirez, Oliver Popp, James D. Sutherland, Sylvie Urbé, Gunnar Dittmar, Michael J. Clague, Ugo Mayor. Quantitative proteomic analysis of Parkin substrates in Drosophila neurons. Molecular Neurodegeneration. 2017; 12 (1):1-19.

Chicago/Turabian Style

Aitor Martinez; Benoit Lectez; Juanma Ramirez; Oliver Popp; James D. Sutherland; Sylvie Urbé; Gunnar Dittmar; Michael J. Clague; Ugo Mayor. 2017. "Quantitative proteomic analysis of Parkin substrates in Drosophila neurons." Molecular Neurodegeneration 12, no. 1: 1-19.

Journal article
Published: 18 January 2017 in Scientific Reports
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Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.

ACS Style

Lucia Pirone; Wendy Xolalpa; Jón Otti Sigurðsson; Juanma Ramirez; Coralia Pérez; Monika Gonzalez; Ainara Ruiz de Sabando; Felix Elortza; Manuel S. Rodriguez; Ugo Mayor; Jesper Olsen; Rosa Barrio; James D. Sutherland. A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation. Scientific Reports 2017, 7, 40756 .

AMA Style

Lucia Pirone, Wendy Xolalpa, Jón Otti Sigurðsson, Juanma Ramirez, Coralia Pérez, Monika Gonzalez, Ainara Ruiz de Sabando, Felix Elortza, Manuel S. Rodriguez, Ugo Mayor, Jesper Olsen, Rosa Barrio, James D. Sutherland. A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation. Scientific Reports. 2017; 7 (1):40756.

Chicago/Turabian Style

Lucia Pirone; Wendy Xolalpa; Jón Otti Sigurðsson; Juanma Ramirez; Coralia Pérez; Monika Gonzalez; Ainara Ruiz de Sabando; Felix Elortza; Manuel S. Rodriguez; Ugo Mayor; Jesper Olsen; Rosa Barrio; James D. Sutherland. 2017. "A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation." Scientific Reports 7, no. 1: 40756.

Book chapter
Published: 10 September 2016 in Methods in Molecular Biology
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Ubiquitination pathways are widely used within eukaryotic cells. The complexity of ubiquitin signaling gives rise to a number of problems in the study of specific pathways. One problem is that not all processes regulated by ubiquitin are shared among the different cells of an organism (e.g., neurotransmitter release is only carried out in neuronal cells). Moreover, these processes are often highly temporally dynamic. It is essential therefore to use the right system for each biological question, so that we can characterize pathways specifically in the tissue or cells of interest. However, low stoichiometry, and the unstable nature of many ubiquitin conjugates, presents a technical barrier to studying this modification in vivo. Here, we describe two approaches to isolate ubiquitinated proteins to high purity. The first one favors isolation of the whole mixture of ubiquitinated material from a given tissue or cell type, generating a survey of the ubiquitome landscape for a specific condition. The second one favors the isolation of just one specific protein, in order to facilitate the characterization of its ubiquitinated fraction. In both cases, highly stringent denaturing buffers are used to minimize the presence of contaminating material in the sample.

ACS Style

Juanma Ramirez; Mingwei Min; Rosa Barrio; Catherine Lindon; Ugo Mayor. Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples. Methods in Molecular Biology 2016, 1449, 193 -202.

AMA Style

Juanma Ramirez, Mingwei Min, Rosa Barrio, Catherine Lindon, Ugo Mayor. Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples. Methods in Molecular Biology. 2016; 1449 ():193-202.

Chicago/Turabian Style

Juanma Ramirez; Mingwei Min; Rosa Barrio; Catherine Lindon; Ugo Mayor. 2016. "Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples." Methods in Molecular Biology 1449, no. : 193-202.