Biology subjects

Ferracci, G.

Publications and source records attributed to Ferracci, G..

6 recordsLinked to original sources

bioRxiv2025

Every Piece of the Puzzle Matters: A Novel Zinc-Binding Site in the Luminal Domain of STIM1 Drives Clustering

STIM1 is pivotal in the tightly regulated mechanism controlling calcium homeostasis in the ER. It is activated by calcium dissociation from its EF-hand domain when ER calcium levels decrease, leading to its interaction with the ORAI channel to initiate calcium influx. Despite advancements in understanding the complex STIM1 machinery, including its domain organization and structural rearrangements upon activation, many aspects of this process remain poorly understood. In this study, we focused on a small conserved region situated upstream to the EF-hand, which has been previously shown to be involved in the modulation of STIM1 by ROS. Our findings reveal that this region binds zinc and plays a pivotal role in STIM1 activation by promoting its clustering, a process essential for the activation of calcium influx. These results revealed the functional importance of this domain and added a crucial piece to the puzzle of how calcium and zinc signaling are interconnected. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/659323v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@16efeborg.highwire.dtl.DTLVardef@1e15e96org.highwire.dtl.DTLVardef@108cad0org.highwire.dtl.DTLVardef@3b58b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Identification and optimization of trans-species reactive TfR1-binding VHH as tools for drug delivery across the blood brain barrier

The blood-brain barrier (BBB) is a major obstacle for delivering therapeutic agents to the central nervous system (CNS), posing significant challenges for treating neurological disorders. Among current strategies to improve brain drug exposure, hijacking physiological pathways involved in receptor-mediated transcytosis (RMT) has emerged as a promising strategy. While targeting transferrin receptor 1 (TfR1) is widely explored, many TfR1-antibodies lack cross-species reactivity, limiting translational development. In the present study, we identified and characterized camelid-derived single-domain antibodies (VHHs) with robust cross-reactivity to rodent, rhesus monkey, and human TfR1. Epitope mapping of the VHH revealed a novel binding site at the interface of the TfR1 dimer. When fused to a human IgG1 Fc domain, these VHHs, as monomers or homodimers, were efficiently internalized by engineered CHO cells and brain endothelial cells expressing TfR1 from different species. Systemic administration of VHH-Fc constructs in mice demonstrated significantly improved brain uptake compared to irrelevant controls. Functional delivery was confirmed using neurotensin (NT)-induced hypothermia, and we established correlations between in vivo effects and TfR1 VHH binding properties determined by surface plasmon resonance. Notably, efficient BBB transcytosis was associated with intermediate affinity and rapid dissociation rates. Engineered variants maintained favorable cross-species binding, including similar affinities to human and non-human primate TfR1, facilitating translational studies. The cross-reactive anti-TfR1 VHHs we developed offer a versatile and modular platform for CNS drug delivery and hold promise as molecular shuttles for transporting therapeutic agents across the BBB. Our work establishes a robust foundation for developing next-generation brain-targeted biotherapeutics, including peptides, enzyme replacement therapies, antibody-based treatments for neurodegenerative diseases, and oligonucleotide delivery for CNS disorders, enabling seamless translation from preclinical to clinical applications.

neuroscience↗

bioRxiv2024

A peptide-neurotensin conjugate that crosses the blood-brain barrier induces pharmacological hypothermia associated with anticonvulsant, neuroprotective and anti-inflammatory properties following status epilepticus in mice

Preclinical and clinical studies show that mild to moderate hypothermia is neuroprotective in sudden cardiac arrest, ischemic stroke, perinatal hypoxia/ischemia, traumatic brain injury and seizures. Induction of hypothermia largely involves physical cooling therapies, which induce several clinical complications, while some molecules have shown to be efficient in pharmacologically induced hypothermia (PIH). Neurotensin (NT), a 13 amino-acid neuropeptide that regulates body temperature, interacts with various receptors to mediate its peripheral and central effects. NT induces PIH when administered intracerebrally. However, these effects are not observed if NT is administered peripherally, due to its rapid degradation and poor passage of the blood brain barrier (BBB). We conjugated NT to peptides that bind the low-density lipoprotein receptor (LDLR) to generate "vectorized" forms of NT with enhanced BBB permeability. We evaluated their effects in epileptic conditions following peripheral administration. One of these conjugates, VH-N412, displayed improved stability, binding potential to both the LDLR and NTSR-1, rodent/human cross-reactivity and improved brain distribution. In a mouse model of kainate (KA)-induced status epilepticus (SE), VH-N412 elicited rapid hypothermia associated with anticonvulsant effects, potent neuroprotection and reduced hippocampal inflammation. VH-N412 also reduced sprouting of the dentate gyrus mossy fibers and preserved learning and memory skills in the treated mice. In cultured hippocampal neurons, VH-N412 displayed temperature-independent neuroprotective properties. To the best of our knowledge, this is the first report describing the successful treatment of SE with PIH. In all, our results show that vectorized NT may elicit different neuroprotection mechanisms mediated either by hypothermia and/or by intrinsic neuroprotective properties.

neuroscience↗

bioRxiv2023

LDLR-mediated targeting and productive uptake of siRNA-peptide ligand conjugates in vitro and in vivo

siRNAs have become one of the most promising therapeutic agents because of their specificity and their potential to modulate the expression of gene-related diseases. Any gene of interest can be potentially up or down-regulated, making RNA-based technology the healthcare breakthrough of our era. However, the functional and specific delivery of siRNAs into tissues of interest and into the cytosol of target cells remains highly challenging, mainly due to the lack of efficient and selective delivery systems. Among the variety of carriers for siRNA delivery, peptides have become essential candidates because of their high selectivity, stability and conjugation versatility. Here, we describe the development of molecules encompassing siRNAs against SOD1, conjugated to peptides that target the LDLR, and their biological evaluation both in vitro and in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/526778v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@11146borg.highwire.dtl.DTLVardef@1537ee2org.highwire.dtl.DTLVardef@af67b5org.highwire.dtl.DTLVardef@1c1d7ec_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG

biochemistry↗

bioRxiv2021

The protein-binding pocket of Botulinum neurotoxin B accommodates a preassembled synaptotagmin / ganglioside complex

Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.

pharmacology and toxicology↗

bioRxiv2021

Sequential binding of zinc triggers tau aggregation

Tau protein has been extensively studied due to its key roles in microtubular cytoskeleton regulation and in the formation of aggregates found in some neurodegenerative diseases. Recently it has been shown that zinc is able to induce tau aggregation by interacting with several binding sites. However, the precise location of these sites and the molecular mechanism of zinc-induced aggregation remain unknown. Here we used Nuclear Magnetic Resonance (NMR) to identify zinc binding sites on hTau40 isoform. These experiments revealed three distinct zinc binding sites on tau, located in the N-terminal part (H14, H32, H94, and H121), the repeat region (H299, C322, H329 and H330) and the C-terminal part (H362, H374, H388 and H407). Further analysis enabled us to show that the C-terminal and the N-terminal sites are independent of each other. Using molecular simulations, we modeled the structure of each site in a complex with zinc. Given the clinical importance of zinc in tau aggregation, our findings pave the way for designing potential therapies for tauopathies. HighlightsO_LIZinc is known to induce tau aggregation in neurodegenerative diseases C_LIO_LIZinc binding locations and mechanism are not yet clear C_LIO_LIUsing NMR we localized 3 zinc binding site on tau C_LIO_LIBy molecular simulations, we proposed a modeled structure of each site C_LIO_LIOur findings pave the way for designing potential therapies for tauopathies C_LI

molecular biology↗