Biology subjects

Lecomte, S.

Publications and source records attributed to Lecomte, S..

6 recordsLinked to original sources

bioRxiv2026

Dysregulation of cAMP-PKA-mediated mechanisms disrupts basal synaptic function in the absence of presynaptic FMRP

The regulation of presynaptic mechanisms by the fragile X messenger ribonucleoprotein (FMRP) remains incompletely understood despite their potential importance to synaptic dysfunction reported in Fragile X syndrome. We combined post-developmental, cell-specific deletion of FMRP in dentate granule cells of the hippocampus with optogenetics, whole-cell patch-clamp electrophysiology and super resolution imaging. The selective loss of FMRP in DG cells did not alter their intrinsic excitability. The amplitude of synaptic currents was reduced at the output mossy fiber synapses in the absence of presynaptic FMRP, whereas short-term facilitation remained intact. We demonstrate that basal synaptic activity is regulated by PKA at mature mossy fiber synapses, and our results indicate alterations in cAMP-PKA signaling and a potential reduction in the readily releasable pool size in mossy fiber boutons lacking FMRP. Presynaptic FMRP is therefore required for efficient basal neurotransmission at mature mossy fiber synapses and likely acts through cAMP-PKA-dependent signaling pathway. Significance statementThe fragile X messenger ribonucleoprotein (FMRP) has been extensively studied as a regulator of postsynaptic mechanisms. However, its physiological functions in presynaptic terminals remain incompletely understood. Using post-developmental, cell-specific deletion of FMRP, we show that presynaptic FMRP is required to maintain efficient basal neurotransmission at hippocampal mossy fiber synapses and identify cAMP-PKA signaling as a potential mechanism for this effect. Taken together, these results highlight a physiological role for presynaptic FMRP during basal synaptic activity and demonstrate how time-controlled gene-editing approaches via viral infection can distinguish primary molecular functions from developmental compensations, thereby shedding new light on both the presynaptic function of FMRP and the pathophysiology of Fragile X syndrome. HighlightsO_LIPresynaptic loss of FMRP reduces basal synaptic activity at hippocampal Mf synapses. C_LIO_LIBasal synaptic activity is regulated by PKA in adult Mf synapses. C_LIO_LIAC-cAMP-PKA pathway is impaired during basal synaptic activity in the absence of presynaptic FMRP. C_LIO_LIRRP size is very likely reduced in AZs at Mf terminals. C_LI

neuroscience↗

bioRxiv2026

Non-fibrillar prion protein oligomers transmit structural information during early assembly

The prion paradigm is founded on the transmission of structural information from aggregated assemblies to soluble protein substrates, a process classically attributed to fibril-end-mediated templating. Whether non-fibrillar assemblies or transient oligomeric states can also participate in folding information transfer remains unclear. Here, using recombinant prion protein (PrP), rational mutagenesis, hetero-oligomerization assays, arrested reaction conditions, and single-particle atomic force microscopy coupled to infrared nano-spectroscopy (AFM-IR), we examine the earliest stages of PrP assembly from the perspective of folding information transmission. We show that polymerization-defective PrP variants can be incorporated into oligomeric assemblies through structural complementation, in which folding information supplied by wild-type PrP restores their assembly competence. Under arrested reaction conditions, transient polymerization-competent conformers further contribute to folding information transfer at subcritical concentrations. Domain-resolved analyses reveal a modular oligomeric architecture in which a {beta}-sheet-rich B domain constitutes the primary scaffold for folding information transfer. Preformed O1 oligomers then act as autonomous conformational templates that promote mutant incorporation and undergo hierarchical condensation through accretion of a structurally distinct E domain. Together, these findings demonstrate that non-fibrillar PrP oligomers and transient assembly intermediates can store and transmit folding information and may function as oligomer-based secondary nucleation platforms, expanding the conceptual framework of prion assembly beyond fibril-end elongation alone.

biochemistry↗

bioRxiv2024

Characterization of widely conserved novel pigment production in Bacillus subtilis species

Bacillus subtilis is widely studied in the microbial secondary metabolite (SM) field due to its rich variety of important natural products and genetic tractability. However, identification of novel SMs and their biosynthetic gene cluster (BGCs) has become increasingly difficult, especially in Bacilli, as the tools for screening and genome mining are dependent on clear function or similarity to already known BGCs. Pigments are SMs identified by their absorption of visible light, resulting in a certain color perceived by our eyes at sufficient concentrations. Thereby, pigments provide the evidence of a BGC without knowing the sequence or function. Expanding the known repertoire of SM BGCs with novel BGCs will further reinforce identification of a broader set of BGCs by mining tools such as antiSMASH. Here, we study a pigment observed in B. subtilis soil isolate MB9_B4 on certain media. We characterize the conditions where this pigment is produced and identify the corresponding BGC using a comparative genomic approach exploiting our strain collection containing other isolates with pigment production ability. The responsible BGC carried several genes, which were annotated as parts of the tryptophan biosynthesis pathway, possibly originating from a duplication and divergence of an originally primary metabolism. Identification of the pigment gene cluster additionally lead to the discovery of additional pigment BGC carrier B. subtilis isolates, some of which were described at the earliest in 1896 under the name Bacillus aterrimus, with a name referring to a dark pigmentation (the Latin "aterrimus" meaning very black). In addition, we employed solid-state nuclear magnetic resonance and Fourier transform infrared spectroscopies to characterize the chemical groups of the pigment. This study describes the chemical and biological features of a new class of SM BGC, which we hope will serve to improve the current BGC discovery pipelines in Bacilli.

microbiology↗

bioRxiv2024

N-formylation modifies membrane damage associated to PSMα3 interfacial fibrillation

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins 3 (PSM3) peptides, able to self-assemble into amyloid-like cross- fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSM3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining Atomic Force Microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSM3 alters its interactions with model membranes of controlled lipid composition, without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSM3 - membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSM3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains, only in the fluid-phase of membranes, and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSM3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation, and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSM3 with specific membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.

biophysics↗

bioRxiv2021

Structural dissection of the first events following membrane binding of the islet amyloid polypeptide

Amyloid forming proteins are involved in many pathologies and often belong to the class of intrinsically disordered proteins. One of these proteins is the islet amyloid polypeptide (IAPP), which is the main constituent of the amyloid fibrils found in the pancreas of type 2 diabetes patients. The molecular mechanism of IAPP-induced cell death is not yet understood, however it is known that cell membrane plays a dual role, being a catalyst of IAPP aggregation and the target of IAPP toxicity. Using FTIR spectroscopy, transmission electron microscopy, and molecular dynamics simulations we investigate the very first molecular steps following IAPP binding to a lipid membrane. In particular, we assess the combined effects of the charge state of amino-acid residue 18 and the IAPP-membrane interactions on the structures of monomeric and aggregated IAPP. Both our experiments and simulations reveal distinct IAPP-membrane interaction modes for the various IAPP variants. Membrane binding causes IAPP to fold into an amphipathic helix, which in the case of H18K- and H18R-IAPP can easily insert into the membrane. For all IAPP variants but H18E-IAPP, the membrane-bound -helical structure is an intermediate on the way to IAPP amyloid aggregation, while H18E-IAPP remains in a stable helical conformation. The fibrillar aggregates of wild-type IAPP and H18K-IAPP are dominated by an antiparallel {beta}-sheet conformation, while H18R- and H18A-IAPP exhibit both antiparallel and parallel {beta}-sheets as well as amorphous aggregates. In summary, our results emphasize the importance of residue 18 for the structure and membrane interaction of IAPP. This residue is thus a good target for destabilizing amyloid fibrils of IAPP and inhibit its toxic actions by possible therapeutic molecules.

biophysics↗

bioRxiv2019

Ionpair-π interactions favor cell penetration of arginine/tryptophan-rich cell-penetrating peptides

Cell-penetrating peptides (CPPs) internalization can occur both by endocytosis and direct translocation through the cell membrane. These different entry routes suggest that molecular partners at the plasma membrane, phospholipids or glycosaminoglycans (GAGs), bind CPPs with different affinity or selectivity. The analysis of sequence-dependent interactions of CPPs with lipids and GAGs should lead to a better understanding of the molecular mechanisms underlying their internalization. CPPs are short sequences generally containing a high number of basic arginines and lysines and sometimes aromatic residues, in particular tryptophans. Tryptophans are crucial residues in membrane-active peptides, because they are important for membrane interaction. Membrane-active peptides often present facial amphiphilicity, which also promote the interaction with lipid bilayers. To study the role of Trp and facial amphiphilicity in cell interaction and penetration of CPPs, a nonapeptide series containing only Arg, Trp or D-Trp residues at different positions was designed. Our quantitative study indicates that to maintain/increase the uptake efficiency, Arg can be advantageously replaced by Trp in the nonapeptides. The presence of Trp in oligoarginines increases the uptake in cells expressing GAGs at their surface, when it only compensates for the loss of Arg and maintains similar peptide uptake in GAG-deficient cells. In addition, we show that facial amphiphilicity is not required for efficient uptake of these nonapeptides. Thermodynamic analyses point towards a key role of Trp that highly contributes to the binding enthalpy of complexes formation. Density functional theory (DFT) analysis highlights that salt bridge-{pi} interactions play a crucial role for the GAG-dependent entry mechanisms.

biochemistry↗