Biology subjects

Barrio, R.

Publications and source records attributed to Barrio, R..

6 recordsLinked to original sources

bioRxiv2025

Loss of UFMylation supports prostate cancer metastasis and rewires cell metabolism towards hexosamine biosynthesis

The acquisition of metastatic features in tumor cells encompasses genetic and non-genetic adaptation, including reprogramming of cellular metabolism. Here we show that loss of UFMylation reroutes glucose metabolism, promotes invasive capacity and supports prostate cancer metastasis. Through transcriptome-based bioinformatics analysis, we identified a reduction in the ubiquitin-like modifier UFM1 and its ligase UFL1 in metastatic prostate cancer. We demonstrate that loss of UFMylation results in enhanced cancer cell dissemination and a switch from cellular proliferation to invasion. Using biotin-based proteomics, we identified phosphofructokinase (PFKAP) as an unprecedented UFMylation substrate. Consistent with UFMylation playing a role in the regulation of phosphofructokinase activity, loss of UFMylation reduced glucose metabolism in favour of hexosamine biosynthesis, which resulted in elevated glycosylation of proteins relevant for cell invasion. These results reveal a role for UFMylation in the regulation of phosphofructokinase and glucose metabolism to support prostate cancer metastasis.

cancer biology↗

bioRxiv2025

Cullin-RING ligase BioE3 reveals molecular-glue-induced neosubstrates and rewiring of the endogenous Cereblon ubiquitome

BackgroundThe 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. MethodsBioE3 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, adding 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 which ubiquitination changes significantly upon treatment was done using two-sided Students t-test. Orthogonal validations were performed by histidine pull-down, GFP-trap and computational modelling. ResultsHere we demonstrate that BioE3 is suitable for the multi-protein complex Cullin-RING E3s ligases (CRLs), the most utilized by targeted protein degradation strategies. Choosing 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. ConclusionsThe ability of BioE3 to detect and compare both substrates and neosubstrates, as well as how substrates change in response to treatments, will facilitate both target and off-target identifications and offer a broader characterization and validation of targeted protein degradation degraders, like molecular glues and PROTACs.

cell biology↗

bioRxiv2023

BioE3 enables the identification of bona fide targets of E3 ligases

The post-translational modification of proteins by ubiquitination is a highly regulated process that involves a dynamic, three-step enzymatic cascade, where more than 600 E3 ligases play a critical role in recognizing specific substrates for modification. Separating bona fide targets of E3s from E3-interacting proteins remains a major challenge in the field. In this study, we present BioE3, a novel approach for identifying substrates of ubiquitin-like (UbL) E3 ligases of interest. Using BirA-E3 ligase fusion proteins and bioUbLs, the method facilitates site-specific biotinylation of UbL-modified substrates of particular E3s for proteomic identification. We demonstrate that the BioE3 system can identify both known and novel targets of two RING-type ubiquitin E3 ligases: RNF4, known to be involved in DNA damage response and the regulation of PML nuclear bodies, and MIB1, implicated in endocytosis, autophagy, and centrosomal protein homeostasis. We further show the versatility of BioE3 by identifying targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, we show that BioE3 works with HECT-type E3 ligases and identify novel targets of NEDD4 involved in vesicular trafficking. BioE3 is a powerful tool that enables identification of bona fide substrates of UbL E3 ligases and how they change with chemical perturbations. BioE3 may also be applicable for UbLs beyond Ub and SUMO, as well as other E3 ligase classes. The resulting knowledge can shed light on the regulation of cellular processes by the complex UbL network and provide information useful for strategies such as targeted protein degradation (TPD), advancing our understanding of fundamental biological mechanisms and their applications.

cell biology↗

bioRxiv2020

Identification of proximal SUMO-dependent interactors using SUMO-ID

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 developed 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 allowed us to identify novel interactors of SUMOylated SALL1, a less characterized SUMO substrate. 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.

biochemistry↗

bioRxiv2019

LUZP1, a novel regulator of primary cilia and the actin cytoskeleton, is altered in Townes-Brocks Syndrome

Primary cilia are sensory organelles that are crucial for cell signaling during development and organ homeostasis. Cilia arise from the centrosome and their formation is governed by numerous regulatory factors. We show that the leucine-zipper protein LUZP1 localizes to the pericentriolar material and actin cytoskeleton. Using TurboID proximity labeling and pulldowns, LUZP1 associates with factors linked to centrosome and actin filaments. Loss of LUZP1 reduces F-actin levels, facilitating ciliogenesis and altering Sonic Hedgehog signaling, pointing to a key role in the cytoskeleton-cilia interdependency. Moreover, we show that LUZP1 interacts with a truncated form of the transcription factor SALL1 that causes Townes-Brocks Syndrome. TBS is characterized by digit, heart and kidney malformations and is linked in part to defective cilia. Truncated SALL1 increases the ubiquitin proteasome-mediated degradation of LUZP1. Alteration of LUZP1 levels may be a contributing factor to TBS, suggesting possible therapies using modulators of cilia and cytoskeletal function.

cell biology↗

bioRxiv2018

Integrative analysis of transcriptomic and clinical data uncovers the tumor suppressive activity of MITF in prostate cancer.

The dysregulation of gene expression is an enabling hallmark of cancer. Computational analysis of transcriptomics data from human cancer specimens, complemented with exhaustive clinical annotation, provides an opportunity to identify core regulators of the tumorigenic process. Here we exploit well-annotated clinical datasets of prostate cancer for the discovery of transcriptional regulators relevant to prostate cancer. Following this rationale, we identify Microphthalmia-associated transcription factor (MITF) as a prostate tumor suppressor among a subset of transcription factors. Importantly, we further interrogate transcriptomics and clinical data to refine MITF perturbation-based empirical assays and unveil Crystallin Alpha B (CRYAB) as an unprecedented direct target of the transcription factor that is, at least in part, responsible for its tumor suppressive activity in prostate cancer. This evidence was supported by the enhanced prognostic potential of a signature based on the concomitant alteration of MITF and CRYAB in prostate cancer patients. In sum, our study provides proof-of-concept evidence of the potential of the bioinformatics screen of publicly available cancer patient databases as discovery platforms, and demonstrates that the MITF-CRYAB axis controls prostate cancer biology.

cancer biology↗