Biochemistry, Genetics and Molecular Biology, Cancer Research, Cell Biology, Biotechnology
14
Scopus Publications
Scopus Publications
Defining the reference proteomes for small extracellular vesicles and non-vesicular components Julia Morales-Sanfrutos, Joanes Etxeberria-Ugartemendia, Orhi Barroso-Gomila, Esperanza González, Maria Sendino, Pilar Ximénez-Embún, Fernando García, Eduardo Zarzuela, Juan M. Falcón-Pérez, Héctor Peinado, Javier Muñoz Nature Cell Biology, 2026 Extracellular vesicles (EVs) are key mediators of intercellular communication and promising biomarkers. However, their molecular characterization remains challenging due to the heterogeneity of EV subtypes and co-isolated non-vesicular components. Here we leverage protein correlation profiling along density gradients to systematically analyse over 9,000 proteins in human cancer cell lines and biofluids, providing a rigorous reassessment of virtually all protein constituents associated with small EVs (sEVs) and non-vesicular entities. We show that sEVs primarily incorporate plasma membrane proteins via selective cargo-loading mechanisms, with low inclusion of intraluminal soluble proteins. By contrast, the abundant cytosolic proteins frequently detected in sEV preparations are not encapsulated within vesicles but are externally associated, probably originating from copurifying cellular debris and aggregates. Our work provides a reference resource for understanding the biogenesis, molecular determinants of cargo selection and functional roles of sEVs. Morales-Sanfrutos, Etxeberria-Ugartemendia, Barroso-Gomila and colleagues map the elution profiles of small extracellular vesicles along density gradients from human biofluids and cell lines, distinguishing extracellular vesicle content from co-isolation components.
A guide to mapping ubiquitin and ubiquitin-like E3 ligases to their substrates Laura Merino‐Cacho, Claudia Guinea‐Pérez, Mónica Pozo‐Rodríguez, Sandra Cano‐López, Juanma Ramirez, Orhi Barroso‐Gomila, Ugo Mayor, James D. Sutherland, Rosa Barrio FEBS Journal, 2026 Ubiquitination is a post‐translational modification that plays a key role in the maintenance of protein homeostasis. Ubiquitin is covalently attached to the target proteins through a three‐step enzymatic cascade in which substrate specificity is conferred by the E3 ligases. However, to match more than 600 E3s with their specific substrates is one of the major challenges in the field. The dynamic and reversible nature of ubiquitination requires the development of techniques to systematically address this question. Here we provide a comprehensive overview of the current methodologies used to reveal targets of E3 ligases, discussing their strengths and limitations. This is particularly relevant in light of emerging pharmacological strategies for targeted protein degradation.
Cullin-RING ligase BioE3 reveals molecular-glue-induced neosubstrates and rewiring of the endogenous Cereblon ubiquitome Laura Merino-Cacho, Orhi Barroso-Gomila, Mónica Pozo-Rodríguez, Veronica Muratore, Claudia Guinea-Pérez, Álvaro Serrano, Coralia Pérez, Sandra Cano-López, Ainhoa Urcullu, Mikel Azkargorta, Ibon Iloro, Carles Galdeano, Jordi Juárez-Jiménez, Ugo Mayor, Felix Elortza, Rosa Barrio, James D. Sutherland Cell Communication and Signaling, 2025 Background The specificity of the ubiquitination process is mediated by the E3 ligases. Discriminating genuine substrates of E3s from mere interacting proteins is one of the major challenges in the field. We previously developed BioE3, a biotin-based approach that uses BirA-E3 fusions together with ubiquitin fused to a low-affinity AviTag to obtain a site-specific and proximity-dependent biotinylation of the substrates. We proved the suitability of BioE3 to identify targets of RING and HECT-type E3 ligases. Methods BioE3 experiments were performed in HEK293FT and U2OS stable cell lines expressing TRIPZ-bioGEFUb transiently transfected with BirA-cereblon (CRBN). Cells were seeded using biotin-free media, followed later by a short-biotin pulse. We evaluated the applicability of the BioE3 system to CRBN and molecular glues by Western blot and confocal microscopy, blocking the proteasome with bortezomib, inhibiting NEDDylation with MLN4924 and treating the cells with pomalidomide. For the identification of endogenous substrates and neosubstrates we analyzed the eluates of streptavidin pull-downs of BioE3 experiments by LC–MS/MS. Analysis of targets for which ubiquitination changes significantly upon treatment was done using two-sided Student’s t-test. Orthogonal validations were performed by histidine pull-down, GFP-trap and computational modelling. Results Here we demonstrate that BioE3 is suitable for the multi-protein complex Cullin-RING E3s ligases (CRLs), the most utilized E3-type for targeted protein degradation (TPD) strategies. Using CRBN as proof of concept, one of the substrate receptors of CRL4 E3 ligase, we identified both endogenous substrates and novel neosubstrates upon pomalidomide treatment, including CSDE1 which contains a G-loop motif potentially involved in the binding to CRBN in presence of pomalidomide. Importantly, we observed a major rearrangement of the endogenous ubiquitination landscape upon treatment with this molecular glue. Conclusions The ability of BioE3 to detect and compare both substrates and neosubstrates, as well as how substrates change in response to treatments, will facilitate both on-target and off-target identifications and offer a broader characterization and validation of TPD compounds, like molecular glues and PROTACs.
Biotin-Based Strategies to Explore the World of Ubiquitin and Ubiquitin-Like Modifiers Laura Merino‐Cacho, Orhi Barroso‐Gomila, Sandra Hernández‐Sánchez, Juanma Ramirez, Ugo Mayor, James D. Sutherland, Rosa Barrio Chembiochem, 2024 A complex code of cellular signals is mediated by ubiquitin and ubiquitin‐like (Ub/UbL) modifications on substrate proteins. The so‐called Ubiquitin Code specifies protein fates, such as stability, subcellular localization, functional activation or suppression, and interactions. Hundreds of enzymes are involved in placing and removing Ub/UbL on thousands of substrates, while the consequences of modifications and the mechanisms of specificity are still poorly defined. Challenges include rapid and transient engagement of enzymes and Ub/UbL interactors, low stoichiometry of modified versus non‐modified cellular substrates, and protease‐mediated loss of Ub/UbL in lysates. To decipher this complexity and confront the challenges, many tools have been created to trap and identify substrates and interactors linked to Ub/UbL modification. This review focuses on an assortment of biotin‐based tools developed for this purpose (for example BioUbLs, UbL‐ID, BioE3, BioID), taking advantage of the strong affinity of biotin‐streptavidin and the stringent lysis/washing approach allowed by it, paired with sensitive mass‐spectrometry‐based proteomic methods. Knowing how substrates change during development and disease, the consequences of substrate modification, and matching substrates to particular UbL‐ligating enzymes will contribute new insights into how Ub/UbL signaling works and how it can be exploited for therapies.
BioE3 identifies specific substrates of ubiquitin E3 ligases Orhi Barroso-Gomila, Laura Merino-Cacho, Veronica Muratore, Coralia Perez, Vincenzo Taibi, Elena Maspero, Mikel Azkargorta, Ibon Iloro, Fredrik Trulsson, Alfred C. O. Vertegaal, Ugo Mayor, Felix Elortza, Simona Polo, Rosa Barrio, James D. Sutherland Nature Communications, 2023 Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.
Identification of proximal SUMO-dependent interactors using SUMO-ID Orhi Barroso-Gomila, Fredrik Trulsson, Veronica Muratore, Iñigo Canosa, Laura Merino-Cacho, Ana Rosa Cortazar, Coralia Pérez, Mikel Azkargorta, Ibon Iloro, Arkaitz Carracedo, Ana M. Aransay, Felix Elortza, Ugo Mayor, Alfred C. O. Vertegaal, Rosa Barrio, James D. Sutherland Nature Communications, 2021 The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.
SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes Immacolata Giordano, Lucia Pirone, Veronica Muratore, Eukene Landaluze, Coralia Pérez, Valerie Lang, Elisa Garde-Lapido, Monika Gonzalez-Lopez, Orhi Barroso-Gomila, Alfred C. O. Vertegaal, Ana M. Aransay, Jose Antonio Rodriguez, Manuel S. Rodriguez, James D. Sutherland, Rosa Barrio Frontiers in Cell and Developmental Biology, 2021 Development is orchestrated through a complex interplay of multiple transcription factors. The comprehension of this interplay will help us to understand developmental processes. Here we analyze the relationship between two key transcription factors: CBX4, a member of the Polycomb Repressive Complex 1 (PRC1), and SALL1, a member of the Spalt-like family with important roles in embryogenesis and limb development. Both proteins localize to nuclear bodies and are modified by the small ubiquitin-like modifier (SUMO). Our results show that CBX4 and SALL1 interact in the nucleoplasm and that increased SALL1 expression reduces ubiquitination of CBX4, enhancing its stability. This is accompanied by an increase in the number and size of CBX4-containing Polycomb bodies, and by a greater repression of CBX4 target genes. Thus, our findings uncover a new way of SALL1-mediated regulation of Polycomb bodies through modulation of CBX4 stability, with consequences in the regulation of its target genes, which could have an impact in cell differentiation and development.
The role of SUMOylation during development Ana Talamillo, Orhi Barroso-Gomila, Immacolata Giordano, Leiore Ajuria, Marco Grillo, Ugo Mayor, Rosa Barrio Biochemical Society Transactions, 2020
