Biology subjects

Merino-Cacho, L.

Publications and source records attributed to Merino-Cacho, L..

3 recordsLinked to original sources

bioRxiv2025

Monocarboxylate transporter 2 is required for the maintenance of myelin and axonal integrity by oligodendrocytes

Neurodegenerative pathologies including multiple sclerosis (MS) are consistently associated with energy deficit in the central nervous system (CNS). This might directly impact myelinating oligodendrocytes as these are particularly vulnerable to metabolic insults. Importantly, oligodendroglial dysfunction and myelin alterations occur in most, if not all neurodegenerative diseases, and are associated with axonal pathology/loss. Thus, elucidating metabolic mechanisms required for oligodendroglial myelin maintenance and axonal support might be crucial to identify therapeutic targets to achieve neuroprotection. While monocarboxylates are important energy fuels for the CNS, their role in myelinating oligodendrocyte function remains unclear. Here we show that, just like neurons, myelinating oligodendrocytes express high affinity monocarboxylate transporter 2 (MCT2) both in mice and humans, which is downregulated in progressive MS. While deletion of MCT2 in mouse oligodendrocytes did not affect the survival of these cells, it resulted in downregulation of lipid synthesis-associated enzymes and failure of myelin maintenance. Moreover, axonal upregulation of lactate dehydrogenase A concomitant with axonal damage was observed but could be alleviated by ketogenic diet. We conclude that oligodendroglial MCT2 is required for myelin maintenance and axonal support, which becomes altered in progressive MS, but may be compensated for by specific metabolic therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/632306v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@2b7b22org.highwire.dtl.DTLVardef@a111fdorg.highwire.dtl.DTLVardef@a410f5org.highwire.dtl.DTLVardef@1556e2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

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↗