Biology subjects

Vanni, S.

Publications and source records attributed to Vanni, S..

At least 19 recordsLinked to original sources

bioRxiv2026

A new class of lipid transfer proteins is required for the recycling of lipids from the P. falciparum digestive vacuole

Malaria parasites endocytose large quantities of hemoglobin from the host erythrocyte, a process critical for parasite survival, leading to extensive membrane internalization. While hemoglobin degradation in the digestive vacuole (DV) is well studied, how the parasite deals with the membranes arriving within the DV is unknown. Here we identified PfTUPA, a previously uncharacterized lipid transfer protein in the DV membrane that is needed for this function. PfTUPA contains a soluble TULIP-like lipid transport domain exposed to the DV lumen and a transmembrane lipid transfer domain of bacterial origin (PqiA) in the DV membrane. Structural comparisons revealed proteins with various PqiA and TULIP-like domain combinations across distant eukaryotic clades, indicating this is a frequent functional partnership. Hence, PfTUPA belongs to a new class of eukaryotic lipid transfer proteins that in malaria parasites is needed for a key function of its biology.

microbiology↗

bioRxiv2026

Capabilities, specificity gaps and training-data dependence of AlphaFold3 across diverse application areas

Structure prediction models have moved from single proteins to assemblies that include diverse biomolecules and their modifications. AlphaFold3 (AF3) and related models extended structural modelling via an all-atom framework, opening many new potential applications in structural biology. We evaluate how well the new capabilities of AF3 translate into application tasks in diverse areas: prediction of ubiquitinated protein structures, T-cell receptor (TCR)-epitope recognition, antibody-antigen complexes, protein-RNA and protein-lipid interactions. We find that, while AF3 can perform well in favourable settings, this performance is uneven across applications. In RNA-target predictions, the model confidence fails to separate genuine from decoy interaction partners and in several tasks accuracy depends on the presence of related complexes in the training set. Taken together, our assessment is more cautious than for AF2, whose gains in modelling monomers and complexes were clear and broadly generalisable. AF3s extension to new biomolecule types shows less consistent performance and generalisation. AF3 can be a powerful tool for hypothesis generation and prioritisation, but its predictions and use of confidence metrics will depend strongly on the specific application area and must be interpreted with respect to training-set overlap. We expect that the benchmarks provided here will serve for testing of future developments in the structure prediction field.

bioinformatics↗

bioRxiv2026

Regulated conformational transitions in seipin define a functional ER–lipid droplet interface

Lipid droplets (LDs) are key organelles in cellular lipid homeostasis that form at the endoplasmic reticulum (ER) through a sequence of membrane rearrangements. While seipin emerged as an essential protein complex for LD biogenesis, how seipin-mediated LD formation proceeds beyond the initial step of neutral lipid nucleation remains unknown. Using a combination of in vitro and in-cell cryogenic electron microscopy (cryo-EM), ultrastructural expansion microscopy, molecular simulations and tailored genetic perturbations, we show that the seipin transmembrane domains undergo large-scale conformational rearrangements that define the architecture of the ER-LD interface and enable LD growth. Cryo-EM of purified Xenopus seipin revealed coexistence of two states: a compact "closed" conformation, consistent with early LD biogenesis, and an "open" conformation in which the transmembrane helices splay out laterally. Molecular dynamics simulations indicate that this open state induces local membrane curvature and promotes triacylglycerol accumulation. We identify conserved flexible linkers between the seipin luminal and transmembrane regions that act as mechanical hinges, enabling this conformational transition. We demonstrate that mutations in these hinge regions hinder seipin opening and affect LD formation in yeast and human cells. Analysis of native ER-LD contacts in human cells using light microscopy and cryo-electron tomography confirms that the seipin complex opens to establish stereotypical ~21-nm necks connecting the ER bilayer and LD monolayer. Moreover, we identify the liver-enriched microprotein SMLR1 as an inhibitor of this seipin conformational transition, providing a regulatory mechanism for seipin-dependent lipid storage in a tissue-specific manner. Together, these data establish seipin opening as a key structural rearrangement at the ER-LD interface that is essential for LD biogenesis and growth.

Cell Biology↗

bioRxiv2026

Substrate-dependent oligomerization modulates DGAT1 activity and subcellular localization

Lipid Droplets (LDs) are ubiquitous organelles that are responsible for intracellular energy storage, in the form of highly esterified lipids such as triglycerides (TGs) and sterol esters. LDs emerge from and engage in stable contact sites with the endoplasmic reticulum (ER), where TG biosynthesis takes place by the action of the acyltransferase DGAT1. Despite the recent cryo-EM determination of the human dimeric structure of DGAT11,2, many aspects of the mechanism underlying TG synthesis in the ER remain unclear. Using a combination of molecular dynamics (MD) simulations, biochemical reconstitutions and fluorescence microscopy in live cells, we characterize several steps of DGAT1 molecular mechanism. We found that DAG preferentially enters the catalytic pocket of DGAT1 from the ER luminal leaflet, via a pathway that involves several conserved residues. Each DGAT1 subunit is able to bind multiple DAG molecules, and the presence of DAG promotes the formation of high-order DGAT1 oligomers. DGAT1 displays a preference for curved bilayers in silico, and it preferentially localizes in the ER tubular network, where LD formation is proposed to take place, upon the increase in its natural substrate diacylglycerol (DAG). Overall, our investigations provide a molecular view of how the interplay between protein oligomerization, subcellular localization and substrate biophysical properties modulate DGAT1 enzymatic activity.

cell biology↗

bioRxiv2026

Macaque retina simulator

The primate retina dissects visual scenes into multiple retinocortical streams. The most numerous retinal ganglion cell (GC) types, midget and parasol cells, are further divided into ON and OFF subtypes. These four GC populations have anatomical and physiological asymmetries, which are reflected in the spike trains received by downstream circuits. Computational models of the visual cortex, however, rarely take GC signal processing into account. We have built a macaque retina simulator with the aim of providing biologically plausible spike trains for downstream visual cortex simulations. The simulator is based on realistic sampling density and receptive field size as a function of eccentricity, as well as on two distinct spatial and three temporal receptive field models. Starting from data from literature and earlier receptive field measurements, we synthetize distributions for receptive field parameters, from which the synthetic units are sampled. The models are restricted for monocular and monochromatic stimuli and follow data from the temporal hemiretina which is more isotropic. We show that the model patches conform to anatomical data not used in the reconstruction process and characterize the responses with respect to spatial and temporal contrast sensitivity functions. This simulator allows starting from a stimulus video and provides biologically plausible spike trains for the distinct unit types. This supports development of thalamocortical primate model systems of vision. In addition, it can provide a reference for more biophysical retina models. The independent parameters are housed in text files supporting reparameterization for particular macaque data or other primate species. Author summaryVisual environment provides a rich source of information, and the visual system structure and function has been studied for decades in many species, including humans. The most complex data in mammalian species are processed in the cerebral cortex, but to date we are still missing a functioning model of cortical computations. While the earlier anatomical and physiological data describe many details of the visual system, to understand the functional logic we need to numerically simulate the complex interactions within this system. To pave the way for simulating visual cortex computations, we have developed a functioning model for macaque retina. The neuroinformatics comprises a review and re-digitized existing retina data from literature, as well as statistics of earlier macaque receptive field data. Finally, we provide software which brings the collected neuroinformatics to life and allows researchers to convert visual input into biologically feasible spike trains for simulation experiments of visual cortex.

neuroscience↗

bioRxiv2026

Lipid Droplet Targeting Drives Substrate Access during Very Long Chain Fatty Acid Activation by Fat1

Fatty acids, the most abundant building blocks for lipid synthesis, require conjugation with Coenzyme A (CoA) for their incorporation into membrane lipids. Of those, very long chain fatty acids (VLCFAs), the precursors of yeast ceramides, are particularly hydrophobic. Yet it remains unresolved where and how the sole VLCFA-CoA synthetase Fat1 activates them. Here we show that lipid droplets (LDs) are both the site of free VLCFAs storage, as well as their place of activation. Free VLCFAs preferentially partition into LDs in molecular simulations and in vitro reconstitutions, whereas Fat1 targets the LD surface in cells via a N-terminal amphipathic helix. Structural predictions identify an essential hydrophobic cavity in Fat1 that connects the active site to the membrane, suggesting that VLCFAs directly shuttle from the LD core into the enzymatic pocket. Our model explains how the organelle targeting of a fatty acid activator evolved to match the biophysical properties and consequently the subcellular localization of its highly hydrophobic substrate.

biochemistry↗

bioRxiv2026

Molecular insights into bulk lipid transport from structural studies of the bridge-like protein VPS13A complexed with the scramblase XKR1

In eukaryotes, bridge-like lipid-transfer proteins (BLTPs) are central in mediating vesicle-independent lipid transfer between organelles. BLTPs span the cytosolic space between organelles at contact sites, featuring hydrophobic channels for lipids to travel between membranes. How BLTPs cooperate with partner proteins to orchestrate lipid delivery remains mysterious. Here we used cryo-electron microscopy to visualize a complex comprising the prototypical BLTP VPS13A and the plasma membrane localized scramblase XKR1 at near-atomic resolution. VPS13A interacts with XKR1 via its PH-domain, priming VPS13As bridge-like lipid-transfer domain to deliver lipids directly to the cytosolic leaflet of the acceptor membrane. In molecular dynamics simulations, such arrangement allows for robust lipid transfer, accelerated by membrane properties. Newly delivered lipids can then be equilibrated between leaflets of the membrane bilayer by the scramblase, allowing for membrane growth. Mechanistic insights regarding lipid delivery by VPS13A are directly applicable to all VPS13 proteins and all BLTP family members more broadly.

cell biology↗

bioRxiv2025

Molecular basis for cellular compartmentalization by an ancient membrane fission mechanism

The emergence of cell compartmentalization depends on membrane fission to create the endomembrane compartments. In eukaryotes, membrane fission is commonly executed by ESCRT-III, a protein complex conserved in all domains of life. However, whether membrane fission was an ancestral ESCRT-III activity predating eukaryogenesis remains unknown. Here we show that ESCRT-IIIA from Asgard Heimdallarchaeota, the closest archaeal relatives of eukaryotes, performs membrane fission through an N-terminal amphipathic helix, which we term Hofund. In eukaryotes, Hofund is fragmented across ESCRT-IIIA paralogs, and disrupting these regions causes severe fission defect in yeast. Remarkably, Heimdallarchaeota Hofund restores fission when fused to defective eukaryotic paralogs. These findings suggest that ESCRT-III-mediated fission arose before eukaryogenesis and later diversified to support the regulatory complexity of eukaryotic compartmentalization.

cell biology↗

bioRxiv2025

Dystonia-associated Torsins sustain CLCC1 function to promote membrane fusion of the nuclear envelope for NPC biogenesis

DYT1 early-onset dystonia is a severe, incurable disorder of the central nervous system caused by mutations in the gene encoding Torsin1A (Tor1A, DYT1). Torsins are ER-resident AAA+-ATPases implicated in lipid metabolism, nuclear pore complex (NPC) biogenesis, and lipoprotein secretion, yet their molecular function that underlies the disease pathology has remained incompletely understood. Here, we have utilized Drosophila melanogaster and human somatic cells as experimental models to shed light on their mode-of-action. Fly germ cells lacking dTorsin are arrested in development and display defects in the final steps of NPC biogenesis due to a failure in fusion of the inner and outer nuclear membranes. Using proximity labelling of Torsin1A in human cells, we identify the conserved membrane protein chloride channel CLIC-like protein 1 (CLCC1) as a novel Torsin binding partner. Absence of human CLCC1 or its Drosophila homolog dClcc1 phenocopied the membrane fusion defects at NPC assembly sites observed upon Torsin deletion. Furthermore, CLCC1 is enriched at arrested fusion sites, suggesting it to be a candidate for the elusive NE membrane fusogen. Importantly, CLCC1/dClcc1 overexpression is sufficient to rescue NPC biogenesis and developmental defects associated with Torsin-loss-of-function. Taken together, our data suggest that Torsin-regulated CLCC1 activity drives membrane fusion during NPC biogenesis and reveal that modulating CLCC1 expression is a promising therapeutic prospect for DYT1 dystonia.

cell biology↗

bioRxiv2025

The molecular mechanism of lipid uptake by membrane-anchored bridge-like lipid transfer proteins.

Lipid transport by bridge-like lipid transfer proteins (BLTPs) is emerging as a key process in lipid and cellular metabolism in both physiological and pathological conditions. However, the precise mechanism of lipid transport by BLTPs has remained elusive. Here, we use extensive all-atom molecular dynamics simulations to characterize the precise mechanism of lipid transfer into the BLTP hydrophobic cavity from donor membranes. For multiple BLTPs, we observe the ability to extract and solubilize lipids without lipid selectivity, and we identify membrane destabilization as a critical parameter to achieve effective lipid desorption. We rationally design a mutant BLTP with altered ability to destabilize lipid bilayers, and we show that this abolishes lipid desorption in silico and protein function in vivo. Taken together, our data provide an atomic-level description of the mechanism of lipid transport by BLTPs, ultimately suggesting alternative strategies to interfere with their activity.

biophysics↗

bioRxiv2025

A conserved mechanism of membrane fusion in nuclear pore complex assembly

The nuclear pore complex (NPC) forms a large channel that spans the double lipid bilayer of the nuclear envelope and is the central gateway for macromolecular transport between the nucleus and cytoplasm in eukaryotes. NPC biogenesis requires the coordinated assembly of over 500 proteins culminating in the fusion of the inner and outer nuclear membranes. The molecular mechanism of this membrane fusion step that occurs in all eukaryotes is unknown. Here, we elucidate the mechanism by which two paralogous transmembrane proteins, Brl1 and Brr6, mediate membrane fusion in S. cerevisiae. Both proteins form multimeric, ring-shaped complexes with membrane remodeling activity. Brl1 is enriched at NPC assembly sites via a nuclear export sequence and then interacts with Brr6 across the nuclear envelope lumen through conserved hydrophobic loops. Disrupting this interaction blocks fusion and halts NPC assembly. Molecular dynamics simulations suggest that the Brl1-Brr6 complex drives membrane fusion by forming a channel across bilayers that enables lipid exchange. Phylogenetic analyses reveal that Brl1/Brr6 homologues are broadly distributed across eukaryotes, and functional experiments in human cells and D. melanogaster establish CLCC1 as an NPC fusogen in metazoans. Together, our results uncover a novel, conserved mechanism for membrane fusion in eukaryotes.

cell biology↗

bioRxiv2025

A cold-inducible phospholipid--protein interaction in brown fat mitochondria optimizes thermogenic capacity

Cold stress elicits dynamic remodeling of the mitochondrial lipidome in brown adipose tissue (BAT), marked by an increase in arachidonoyl-phosphatidylethanolamine (AA-PE). However, the function of membrane lipid rewiring in thermoregulatory physiology has been a longstanding mystery. Here, we identify LPCAT3 as a cold-regulated O-acyltransferase driving the highly selective accrual of AA-PE in BAT mitochondria. Lipid-based proteomics, molecular dynamics simulations, and bioenergetic analyses reveal that AA-PE partitions at the COX4I1 interface of the Cytochrome c oxidase complex, enhancing electron transport chain (ETC) efficiency. Accordingly, fat-specific Lpcat3-knockout mice have defects in respiratory-dependent BAT thermogenesis and cold tolerance, despite intact {beta}-adrenergic signaling and UCP1 function. Under cold acclimation, Lpcat3-/-BAT exhibits ETC dysfunction and activation of the integrated stress-response. Thus, our study illuminates a cold-regulated lipid-protein interaction as a gating factor in UCP1-dependent thermogenesis.

physiology↗

bioRxiv2025

TMEM170 family proteins are lipid scramblases that physically associate with bridge lipid transporters BLTP1/Csf1

Bulk lipid transport between organelles has been proposed to involve the partnership between bridge lipid transport proteins and membrane-embedded lipid scramblases. However, for almost all BLTPs, such physical association has not been fully described, and, in most cases, the identity of the scramblases is unknown. Here, we identify TMEM170 family proteins as endoplasmic reticulum lipid scramblases that physically interact with BLTP1/Csf1 proteins. This finding opens new avenues to understand the complex mechanism involved in lipid transport at membrane contact sites.

cell biology↗

bioRxiv2025

Pex30-dependent membrane contacts sites maintain ER lipid homeostasis

In eukaryotic cells, communication between organelles and the coordination of their activities depend on membrane contact sites (MCS). How MCS are regulated under the dynamic cellular environment remains poorly understood. Here, we investigate how Pex30, a membrane protein localized to the endoplasmic reticulum (ER), regulates multiple MCS in budding yeast. We show that Pex30 is critical for the integrity of ER MCS with peroxisomes and vacuoles. This requires the Dysferlin domain (DysF) on Pex30 cytosolic tail. This domain binds to phosphatidic acid (PA) both in vitro and in silico, and it is important for normal PA metabolism in vivo . The DysF domain is evolutionarily conserved and may play a general role in PA homeostasis across eukaryotes. We further show that ER-Vacuole MCS requires Pex30 C-terminal Domain of Unknown Function and that its activity is controlled by phosphorylation in response to metabolic cues. These findings provide new insights into the dynamic nature of MCS and their coordination with cellular metabolism.

cell biology↗

bioRxiv2024

Parameters for sensing lipid loosening at the inner nuclear membrane by amphipathic helices

Amphipathic helices (AHs) are ubiquitous protein motifs that modulate targeting to organellar membranes by sensing differences in bulk membrane properties. However, the adaptation between membrane-targeting AHs and the nuclear membrane environment that surrounds the genome is poorly understood. Here, we computationally screened for candidate AHs in a curated list of characterized and putative human inner nuclear membrane (INM) proteins. Cell biological and in vitro experimental assays combined with computational calculations demonstrated that AHs detect lipid packing defects over electrostatics to bind to the INM, indicating that the INM is loosely packed under basal conditions. Membrane tension resulting from hypotonic shock further promoted AH binding to the INM, whereas cell-substrate stretch did not enhance recruitment of membrane tension-sensitive AHs. Together, our work demonstrates the rules driving lipid-protein interactions at the INM, and its implications in the response of the nucleus to different stimuli.

cell biology↗

bioRxiv2024

Dietary control of peripheral adipose storage capacity through membrane lipid remodelling

Complex genetic and dietary cues contribute to the development of obesity, but how these are integrated on a molecular level is incompletely understood. Here, we show that PPAR{gamma} supports hypertrophic expansion of adipose tissue via transcriptional control of LPCAT3, a membrane-bound O-acyltransferase that enriches diet-derived omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in the phospholipidome. In high-fat diet-fed mice, lowering membrane n-6 PUFA levels by adipocyte-specific Lpcat3 knockout (Lpcat3AKO) or by dietary lipid manipulation leads to dysfunctional triglyceride (TG) storage, ectopic fat deposition and insulin resistance. Aberrant lipolysis of stored TGs in Lpcat3AKO adipose tissues instigates a non-canonical adaptive response that engages a futile lipid cycle to increase energy expenditure and limit further body weight gain. Mechanistically, we find that adipocyte LPCAT3 activity promotes TG storage by selectively enriching n-6 arachidonoyl-phosphatidylethanolamine at the ER-lipid droplet interface, which in turn favours the budding of large droplets that exhibit greater resistance to ATGL-dependent hydrolysis. Thus, our study highlights the PPAR{gamma}-LPCAT3 pathway as a molecular link between dietary n-6 PUFA intake, adipose expandability and systemic energy balance.

physiology↗

bioRxiv2024

RNA order regulates its interactions with zwitterionic lipid bilayers

RNA-lipid interactions directly influence RNA activity, which plays a crucial role in developing new applications in medicine and biotechnology. However, while specific preferential behaviors between RNA and lipid bilayers have been identified experimentally, their molecular origin remains unexplored. Here we use molecular dynamics simulations to investigate the interaction between RNA and membranes composed of zwitterionic lipids at the atomistic level. Our data reproduce and rationalize previous experimental observations, including that short-chain RNAs rich in guanine have higher affinity for gel-phase membranes compared to RNA sequences rich in other nucleotides and that RNA prefers gel-phase membranes to fluid bilayers. Our simulations reveal that RNA order is a key molecular determinant of RNA-zwitterionic phospholipid interactions. Our data provides a wealth of information at the atomic level that will help accelerating research on RNA-lipid assemblies for task-specific applications such as designing lipid-based nanocarriers for RNA delivery.

biophysics↗

bioRxiv2024

An acylated N-terminus and a conserved loop regulate the activity of the ABHD17 de-acylase

The dynamic addition and removal of long chain fatty acids modulates protein function and localization. The alpha/beta hydrolase domain-containing (ABHD) 17 enzymes remove acyl chains from membrane localized proteins such as the oncoprotein NRas, but how the ABHD17 proteins are regulated is unknown. Here, we used cell-based studies and molecular dynamics simulations to show that ABHD17 activity is controlled by two mobile elements - an S-acylated N-terminal helix and a loop - that flank the substrate-binding pocket. S-acylation at multiple sites buries the helix in the membrane, which allows hydrophobic residues in the loop to interact with the bilayer. This stabilizes the conformation of both helix and loop, alters the conformation of the binding pocket and optimally positions the enzyme for substrate engagement. S-acylation may be a general feature of acyl-protein thioesterases. By providing a mechanistic understanding of how the lipid modification of a lipid-removing enzyme promotes its enzymatic activity, this work contributes to our understanding of cellular S-acylation cycles.

cell biology↗