Biology subjects

Estevez, R.

Publications and source records attributed to Estevez, R..

6 recordsLinked to original sources

bioRxiv2026

Structural basis of selective phospholipid recognition and local membrane deformation by CTL1

The physiological function of the human choline transporter-like protein CTL1 (SLC44A1) remains elusive despite its implication in key pathophysiological processes. Here we combine cryo-electron microscopy, native mass spectrometry, lipidomics, molecular dynamics simulations and functional assays to characterise the molecular basis of phospholipid recognition by CTL1 and explore its potential molecular role. High-resolution structures of CTL1 in both monomeric and dimeric states reveal a broad amphipathic cavity and an extracellular domain that accommodate multiple phospholipid species, identifying phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidic acid as endogenous CTL1 ligands. Molecular dynamics simulations show spontaneous phospholipid recruitment into these conserved binding sites accompanied by membrane thinning, curvature and local phospholipid reorganisation, with membrane deformation being enhanced upon dimerisation. Functional analyses in mammalian cells and in reconstituted proteoliposomes failed to detect CTL1-mediated choline transport, supporting a function distinct from canonical choline uptake. Together, our findings suggest that CTL1 acts as a phospholipid-binding protein capable of locally deforming the membrane and provide a structural framework linking phospholipid recognition to membrane organisation. These results offer new insights into potential mechanisms underlying the altered phospholipid homeostasis associated with CTL1 dysfunction in neurological diseases and cancer.

biophysics↗

bioRxiv2025

Cell-type Specific Extracellular Vesicles in Mouse Brain: Proteomic Signatures Highlight Astrocytic GlialCAM Network and GPCR Enrichment

Extracellular vesicles (EVs) mediate intercellular communication in the central nervous system (CNS) and are emerging as biomarkers of brain health and disease. However, the molecular composition of cell type-specific brain EVs, particularly astrocyte-derived EVs (ADEVs), remains poorly defined. We performed comparative proteomic analysis of neuronal (NDEVs), microglial (MDEVs), and astrocytic (ADEVs) from mouse brain using magnetic immunocapture and LC-MS/MS proteomic profiling. Each EV subtype displayed distinct molecular fingerprints. NDEVs were enriched in synaptic and neurogenesis-related proteins (e.g., APP, SNAP25, GPR158 and BDNF), whereas MDEVs contained immune and phagocytic markers (e.g., TMEM119, CX3CR1, CD11B). Strikingly, the ADEV proteome closely mirrored the recently characterized GlialCAM interactome from leukodystrophy research, encompassing GlialCAM/MLC1 and associated partners involved in ion and water homeostasis (EAAT1/2, AQP4, GJA1), together with GPCRs such as GPRC5B. This overlap suggests that ADEVs encapsulate a molecular scaffold characteristic of astrocytic endfeet, potentially extending their signaling functions to the extracellular space. In conclusion, our study provides a detailed comparative proteomic characterization of brain cell type-specific EVs, revealing that ADEVs contain the GlialCAM/MLC1 network and GPCRs. These findings highlight ADEVs as promising candidates for biomarker development and mechanistic studies of blood-brain barrier integrity and neurodegenerative disorders.

neuroscience↗

bioRxiv2025

Structural insights into a conserved mechanism of choline translocation through CHT

The essential nutrient choline is critical for cellular homeostasis across all domains of life. In humans, choline uptake in cholinergic neurons for its recycling into acetylcholine is mediated by the high-affinity Na-dependent transporter SLC5A7 (also known as CHT1). Prokaryotes also depend on choline as an osmo-protectant and as metabolite, raising the possibility that bacteria also possess choline transporters akin to CHT1. Here, we present a bacterial Na+-dependent choline transporter (sfCHT) with high sequence identity to CHT1. sfCHT transport activity can be blocked by the choline transport inhibitor hemicholinium-3. Cryo-EM structures of Na+- and choline-bound sfCHT reveal a 14 transmembrane helix topology with a LeuT-fold architecture and Na+ coordination geometry similar to CHT1. Captured in an inward-facing conformation, in sfCHT choline is found at a site near the cytoplasmic side. Computational analysis and transport assays reveal local conformational changes along a choline translocation pathway to the cytosolic site. Transport assays with CHT1 variants, carrying substitutions at conserved residues along the proposed translocation pathway in sfCHT, reveal a conserved mechanism of choline transport between the bacterial and human choline transporters.

biophysics↗

bioRxiv2021

Ubr1-induced selective endo-phagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress

The defence mechanisms against endo-lysosomal homeostasis stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) ubiquitin ligase that counteracts proteostasis stress by enhancing cargo selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations increased intracellular Ca2+ and caused endosomal compartment stress by fusion and enlargement. Endosomal protein QC pathway using ubiquitin QC ligases CHIP and Ubr1 with ESCRT-machinery was able to target only a fraction of MLC1-mutants for lysosomal degradation. As a consequence of the endosomal stress, we found an alternative QC route dependent on Ubr1, SQSTM1/p62 and arginylation to bypass MLC1-mutants to endosomal autophagy (endo-phagy). Significantly, this unfolded a general biological endo-lysosomal QC pathway for arginylated Ubr1-SQSTM1/p62 autophagy targets during Ca2+-assault. Conversely, the loss of Ubr1 with the absence of arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1-dependent endo-phagy/autophagy in constitutive and provoked endo-lysosomal proteostasis stress, and link Ubr1 to Ca2+-homeostasis and proteins implicated in various diseases including cancers and brain disorders.

biochemistry↗

bioRxiv2021

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC); this rare type of leukodystrophy is characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes (PvAPs, which along with blood vessels form the gliovascular unit (GVU)). We analyzed the GVU in the Mlc1 knock-out mouse model of MLC. The absence of MLC1 led to an accumulation of fluid in the brain but did not modify the endothelial organization or the integrity of the blood-brain barrier. From P10 onward, the postnatal acquisition of vascular smooth muscle cell contractility was altered, resulting in a marked reduction in arterial perfusion and neurovascular coupling. These anomalies were correlated with alterations in astrocyte morphology, astrocyte polarity and the structural organization of the PvAPs perivascular coverage, and poor intraparenchymal circulation of the cerebrospinal fluid (CSF). Hence, MLC1 is required for the postnatal development and organization of PvAPs and controls vessel contractility and intraparenchymal interstitial fluid clearance. Our data suggest that (i) MLC is a developmental disorder of the GVU, and (ii) PvAP and VSMC maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

neuroscience↗

bioRxiv2018

Glia mediated ionic balance in the stem cell niche is required for the proper proliferation of neurogenic tissues and wiring of neural circuits.

The cellular niche that surrounds stem cells modulates their self-renewal and proliferative properties. In the nervous system glial cells form part of the neural stem cell niche and express a wide variety of ion channels. While the physiological roles of ion channels in glia have been mainly studied in the mature nervous system, the contribution of glial ion channel function in the niche to nervous system development is poorly understood. To gain insight we explored the function of the Drosophila ClC-a chloride channel since its human ortholog CLCN2 is expressed in glial cells and impairment of its function results in neurodevelopmental disorders. We found ClC-a expressed in the niche in cortex glia. These glial cells are in close association with neurogenic tissues and also wrap neuronal cell bodies as they mature away from the niche. Characterization of loss of function ClC-a mutants showed these animals had smaller brains and widespread axon guidance defects. Here we show that ClC-a is required in cortex glia for the proliferation of neuroepithelial cells and neuroblasts, as well as for neuronal survival. We could also relate the axon guidance defects observed in photoreceptors to impaired proliferation in a neuroblast lineage that generates guidepost glial cells essential for this process. Taken together, our results show that ion channels expressed in glia forming part of the neural stem cell niche can have a significant impact on nervous system development. We propose that ion channels can non-autonomously modulate the number of neural progenitors and the progeny of neuroblast lineages, consequently affecting the correct assembly of neural circuits.

developmental biology↗