Biology subjects

Vallese, F.

Publications and source records attributed to Vallese, F..

9 recordsLinked to original sources

bioRxiv2025

Deep Red Blood Cell Proteome Defines the Band 3 N-Terminus Interactome as a Regulator of Hypoxic Adaptation via BLVRB-Dependent S-Nitroso Transfer

Red blood cells (RBCs) have long been regarded as passive oxygen carriers, yet growing evidence reveals a complex, dynamic proteome independent of de novo gene expression. Here, we define the erythrocyte as an oxygen-responsive system organized around a Band 3 (SLC4A1)-centered metabolon. Using deep proteomics of ultra-pure RBCs and cross-linking interactomics, we identify biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that binds the N-terminal cytosolic domain under normoxia and dissociates under hypoxia, when band 3-deoxyhemoglobin interactions increase threefold. This reversible interaction forms an oxygen-sensitive switch coupling structural, redox, and metabolic remodeling. In humanized mice, truncation of the Band 3 N-terminus disrupted glycolytic activation, reduced 2,3-bisphosphoglycerate synthesis, and impaired exercise tolerance despite preserved cardiopulmonary function, establishing the physiological relevance of this module. Population-scale proteome quantitative trait locus (pQTL) analyses revealed coordinated variation of SLC4A1 and BLVRB abundance but minimal association of biliverdin levels with BLVRB genotype, suggesting alternative functions beyond heme catabolism. Mechanistically, BLVRB Cys109 acts as a nitric oxide (NO) relay, trans-nitrosating glycolytic enzymes such as GAPDH at active site Cys152, transiently inhibiting glycolysis. This S-nitrosation-mediated feedback mirrors conserved mechanisms in plants, where GAPDH-SNO redirects carbon flow toward the Calvin-Benson cycle under nitrosative stress, revealing an evolutionary convergence in gas-responsive metabolic control. Collectively, our findings define a Band 3-BLVRB-hemoglobin axis that links oxygen sensing, NO signaling, and redox homeostasis, providing a unifying model for how an anucleate cell achieves environmental adaptability through reversible protein-protein interactions and post-translational chemistry. Graphic abstractIssaian et al. define the most comprehensive proteome of ultra-pure human red blood cells (3,775 proteins) and map the O2-dependent interactome, revealing a Band 3-BLVRB-hemoglobin module that links oxygen sensing to metabolic remodeling via reversible inhibitory S-nitrosation of GAPDH C152. In plants this redirects carbon toward photosynthesis, illustrating a conserved NO-dependent metabolic reprogramming mechanism across oxygen-regulated systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/691178v1_ufig1.gif" ALT="Figure 1"> View larger version (89K): org.highwire.dtl.DTLVardef@122468org.highwire.dtl.DTLVardef@114428dorg.highwire.dtl.DTLVardef@11d40c4org.highwire.dtl.DTLVardef@1870fb7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeep proteomics defines a complete, contamination-free RBC proteome (3,775 proteins) C_LIO_LICross-linking proteomics maps an oxygen-sensitive Band 3-centered interactome C_LIO_LIO2-dependent BLVRB-Band 3 binding regulates metabolism via S-nitrosation of GAPDH C_LIO_LIBand 3 N-terminus is required for hypoxic remodeling and exercise tolerance in vivo C_LI

biochemistry↗

bioRxiv2025

Genetic variation of human G6PD impacts Red Blood Cell transfusion efficacy

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common human enzymopathy, affects 6% of the global population, yet its impact on blood storage and transfusion efficacy remains undefined. We integrated genome-metabolome-proteome analyses of 13,091 blood donors (362 G6PD SNPs), validated in a recalled cohort (n=643), linked donor-recipient databases, humanized mouse models (canonical, African A- [V68M+N126D], Mediterranean [S188F]), and a prospective sickle cell disease study. Common G6PD variants reduced protein abundance, reprogrammed redox metabolism, and increased storage hemolysis. In mice, G6PD-deficient RBCs showed lower post-transfusion recovery, higher oxidative stress, and impaired renal oxygenation. Clinically, recipients of G6PD-deficient units exhibited smaller hemoglobin increments and reduced RBC L{superscript 1}Cr-survival (-8% at 24 h; -12% at 4 weeks). Structural studies revealed kinetic fragility for A- and thermodynamic fragility for Med-, linking genotype to protein instability and transfusion outcome. These findings identify donor G6PD genotype as a determinant of transfusion efficacy, supporting genotype-aware inventory-management strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/689741v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1897489org.highwire.dtl.DTLVardef@1420587org.highwire.dtl.DTLVardef@178e8ddorg.highwire.dtl.DTLVardef@1003b44_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Stomatin encapsulates aquaporin-1 and urea transporter-B in the erythrocyte membrane

Stomatin is a ubiquitous and highly expressed protein in erythrocytes, which associates with cholesterol-rich microdomains in the plasma membrane and is known to regulate the activity of multiple ion channels and transporters, but the structural basis of association with stomatin targets remains unknown. Here we describe high-resolution structures of multiple stomatin complexes with endogenous binding partners isolated from human erythrocyte membranes, revealing that stomatin specifically associates with two membrane proteins involved in water transport and cell volume regulation, aquaporin-1 (AQP-1) and the urea transporter, UT-B (SLC14A1). Together, our results reveal the structural basis of stomatin oligomerization, membrane association, and target recruitment, and identify a putative role for stomatin in the regulation of osmotic balance in the erythrocyte.

biochemistry↗

bioRxiv2025

The vault particle is enclosed by a C13-symmetric cap with a positively charged exterior

Vaults are some of the largest ribonucleoprotein complexes known, and are highly conserved across eukaryotes, but both their function and key details of their architecture remain unclear. While high-resolution structures of the vault shell are available, the architecture and symmetry of the cap at either end of the vault has remained unresolved. Here we present a 2.25 [A] cryo-EM structure of the vault cap, revealing an unexpected 13-fold symmetric arrangement that contrasts with the 39-fold symmetry of the vault body, with each repeating module of the cap formed by an asymmetric homotrimer of adjacent subunits, in which two C-termini remain in the vault interior, and one projects into the cytosol. The center of the cap features an unusual architecture reminiscent of the Stomatin, Flotillin and HflK/C (SPFH) superfamily, consisting of two concentric beta barrels surrounded by an interwoven two-layer stack of alpha-helices, with the innermost barrel forming a 15 [A] aperture connecting the interior of the vault with the cytosol. The vault cap features a positively charged exterior and a negatively charged interior surface, with implications for binding partner recruitment and putative binding of the vault particle to microtubules and lipid rafts. These findings uncover a new facet of vault particle architecture, and have implications for engineering and design of modified vault particles for therapeutic delivery, as well as providing new opportunities for interrogating the functional roles of the vault particle in biological systems.

biophysics↗

bioRxiv2024

Accurate and fast segmentation of filaments and membranes in micrographs and tomograms with TARDIS

Segmentation of macromolecular structures is the primary bottleneck for studying biomolecules and their organization with electron microscopy in 2D/3D - requiring months of manual effort. Transformer-based Rapid Dimensionless Instance Segmentation (TARDIS) is a deep learning framework that automatically and accurately annotates membranes and filaments. Pre-trained TARDIS models can segment electron tomography (ET) reconstructions from both 3D and 2D electron micrographs of cryo and plastic-embedded samples. Furthermore, by implementing a novel geometric transformer architecture, TARDIS is the only method to provide accurate instance segmentations of these structures. Reducing the annotation time for ET data from months to minutes, we demonstrate segmentation of membranes and filaments in over 13,000 tomograms in the CZII Data Portal. TARDIS thus enables quantitative biophysical analysis at scale for the first time. We show this in application to kinetochore-microtubule attachment and viral-membrane interactions. TARDIS can be extended to new biomolecules and applications and open-source at https://github.com/SMLC-NYSBC/TARDIS.

bioinformatics↗

bioRxiv2024

Impact of anionic lipids on the energy landscape of conformational transition in anion exchanger 1 (AE1)

Anion Exchanger 1 (AE1) is an elevator-type membrane transporter that plays a key role in erythrocytes by mediating the exchange of chloride and bicarbonate ions across the membrane, thus participating in acid-base homeostasis. While previous studies have provided structural insights into AE1 and its substrate binding, the conformational transitions and the role of lipid interactions remain elusive. In this study, we utilized cryo-electron microscopy (cryo-EM) to resolve three high resolution structures of distinct conformational states of AE1: two inward-facing (IF1 and IF2) and one outward-facing (OF). Furthermore, uptake assay revealed the modulatory effect of phosphatidylinositol 4,5-bisphosphate (PIP2) lipids on AE1. Molecular dynamics (MD) simulations were conducted on these structures to capture anion binding and determine the anion binding sites in AE1. We then used a combination of advanced enhanced sampling techniques together with system-specific collective variables to study the OF{lrdoublearrow}IF transition in AE1 and provided refined pathways for the process in three different systems: apo, HCO3- -bound, and an AE1 system in which cryo-EM-determined interfacial PIP2 lipids had been removed. The refined pathways were then used to calculate the free energy of the OF{lrdoublearrow}IF transition in AE1 under different conditions. The energies show how substrate binding reduces the transition barrier and, therefore, facilitates the transport. Furthermore, they clearly capture the inhibitory effect of PIP2 lipids at the dimer interface. Furthermore, the simulation results provide a molecular mechanism for this inhibitory effect. These results provide a molecular-level understanding of the mechanistic basis for ion transport in AE1 and the regulatory role of PIP2 on its function.

biophysics↗

bioRxiv2024

Revealing biomolecular structure and motion with neural ab initio cryo-EM reconstruction

Proteins and other biomolecules form dynamic macromolecular machines that are tightly orchestrated to move, bind, and perform chemistry. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) can access the intrinsic heterogeneity of these complexes and are therefore key tools for understanding their function. However, 3D reconstruction of the collected imaging data presents a challenging computational problem, especially without any starting information, a setting termed ab initio reconstruction. Here, we introduce cryoDRGN-AI, a method leveraging an expressive neural representation and combining an exhaustive search strategy with gradient-based optimization to process challenging heterogeneous datasets. Using cryoDRGN-AI, we reveal new conformational states in large datasets, reconstruct previously unresolved motions from unfiltered datasets, and demonstrate ab initio reconstruction of biomolecular complexes from in situ data. With this expressive and scalable model for structure determination, we hope to unlock the full potential of cryo-EM and cryo-ET as a high-throughput tool for structural biology and discovery.

biophysics↗

bioRxiv2022

Architecture of the human erythrocyte ankyrin-1 complex

The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2 and glycophorin A. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.

biochemistry↗

bioRxiv2021

Extremely potent human monoclonal antibodies for the prophylaxis and therapy of Tetanus

Human monoclonal antibodies were used here to study the mechanism of neuron intoxication by tetanus neurotoxin protein toxins and as a safe preventive and therapeutic substitute of hyperimmune sera. By screening memory B cells of immune donors, we selected two monoclonal antibodies specific for tetanus neurotoxin with exceptionally high neutralizing activities, which have been extensively characterized both structurally and functionally. We found that these antibodies interfere with the binding and translocation of the neurotoxin into neurons by interacting with two epitopes, whose definition pinpoints crucial events in the cellular pathogenesis of tetanus. Some mechanistic aspects of tetanus neurotoxin intoxication were revealed, explaining at the same time, the unprecedented neutralization ability of these antibodies. Importantly, these antibodies are exceptionally efficient in preventing experimental tetanus when injected in mice long before the neurotoxin. Moreover, their Fab derivatives neutralize tetanus neurotoxin in post-exposure experiments, suggesting their potential therapeutic use upon intrathecal injection. As such, these human monoclonal antibodies, as well as their Fab derivatives, meet all requirements for being considered for prophylaxis and therapy of human tetanus and are ready for clinical trials.

pathology↗