Biology subjects

Brule, S.

Publications and source records attributed to Brule, S..

7 recordsLinked to original sources

bioRxiv2026

Interlaboratory benchmarking of differential scanning fluorimetry reveals sources of variability in protein thermal stability parameters

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two phase interlaboratory benchmark using a shared VHH sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant reported apparent melting temperatures (Tm) showed good within instrument repeatability but moderate inter instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data acceptance criteria in addition to instrument dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent Tm depended strongly on the fluorescence readout. For this VHH, single wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF derived apparent unfolding enthalpies were more variable than apparent Tm. Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data quality criteria, and well characterised reference materials to monitor instrument dependent offsets.

biophysics↗

bioRxiv2026

Ecological Diversification into Broad Thermal Niches Revealed by Protein Resurrection and Proteome-Wide Ancestral Reconstruction During the Rapid Radiation of Alvinellid Worms

In the deep Pacific and Indian oceans, Alvinellid worms diversified about 100 million years ago to colonize a variety of hydrothermal vent environments. It has been suggested that the last common ancestor of this family was a thermophilic species. However, the evolutionary history of these worms is complex, with putative gene flow among ancestors. In this study, we investigated the early evolution of the family in relation to the diversification of thermal niches. We demonstrated that phylogenetic histories of alvinellid species greatly vary along chromosomes, possibly due to allele introgression between nascent ecotypes. We performed sequence reconstructions of ancestral cytosolic MDH and Cu/Zn SOD under the two most frequent phylogenies encountered along the genome. In silico simulations of folding stability for these two enzymes were highly correlated with their biophysical characterization (micro-calorimetry and differential scanning fluorimetry), and were used to predict the thermostability of thousands of alternatively reconstructed proteins. We further generalized the reconstruction at the proteome scale, taking the amino-acid usage bias as a proxy for folding stability in a new phylogenetic model accounting for amino-acid variations over time. Both approaches agree that the last common ancestors of Alvinellidae gained highly stable proteins, comparable to proteins of present-day thermotolerant species. Our computational protocol allowed us to validate this scenario under multiple phylogenetic hypotheses and different sequence reconstruction models. Considered together, Alvinellidae shed light on how metazoan species diversify in order to colonize different thermal niches, combining adaptive mutation and selection on genetic variants. SignificanceDeep-sea hydrothermal vents are among the most extreme environments on Earth. They, however, represent oases of life for a restricted number of highly specialized species. As such, Alvinellid worms constitute an exceptional family, including highly thermotolerant animals such as the Pompeii worm. Despite their vent endemicity, Alvinellidae thrive under contrasting ecological conditions, particularly regarding temperature. Understanding how this family diversified to colonize broad thermal regimes is of major importance for dissecting the mechanisms by which species adapt in highly unstable environments. Combining experimental protein resurrection, proteome-wide predictions, and new phylogenetic models, we assess the reliability of ancestral sequence reconstructions that represent molecular thermometers to evaluate past environmental conditions.

evolutionary biology↗

bioRxiv2021

Interactions of SARS-CoV-2 protein E with cell junctions and polarity PDZ-containing proteins

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified ten human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, LNX2) and MPP5/Pals1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2 and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4 and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

biochemistry↗

bioRxiv2021

The dengue virus NS1 protein conveys pro-inflammatory signals by docking onto human high-density lipoproteins

The nonstructural NS1 protein is a virulence factor secreted by dengue virus (DENV)-infected cells. NS1 is known to alter the complement system, activate immune cells and perturb endothelial barriers. Here we show that pro-inflammatory signals are triggered by a high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Electron microscopy images of the NS1-HDL complexes show spherical HDL particles with rod-shaped NS1 protrusions on their surface. These complexes are readily detectable in the plasma of hospitalized dengue patients using anti-apolipoprotein A-I (ApoA-I) antibodies specific of the HDL moiety. The functional reprogramming of HDL particles by the NS1 protein as a means to exacerbate systemic inflammation during DENV infection provides a new paradigm linking the human lipoprotein network to dengue pathogenesis.

microbiology↗

bioRxiv2021

Establishment of the family Zarkiaceae (Oscillatoriales, Cyanobacteria) and description of the new marine genera Zarkia (Zarkiaceae, Oscillatoriales) and Romeriopsis (Leptolyngbyaceae, Synechococcales), from northern Portugal

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/439031v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1ed7edaorg.highwire.dtl.DTLVardef@1234542org.highwire.dtl.DTLVardef@97f6cborg.highwire.dtl.DTLVardef@cb8cce_HPS_FORMAT_FIGEXP M_FIG C_FIG The morphology, 16S rRNA gene phylogeny and the 16S-23S rRNA gene ITS secondary structures of three strains of marine Cyanobacteria, isolated from inter- and subtidal environments from north Portugal were studied, resulting in the description of Zarkia subtidalensis gen. et. sp. nov. (Zarkiaceae fam. nov.) and Romeriopsis marina gen. et. sp. nov (Leptolyngbyaceae). No diacritical morphological characters were found either for the new family or for the new genera. The 16S rRNA gene Maximum Likelihood and Bayesian phylogenies supported that Zarkia and Zarkiaceae are members of the Oscillatoriales, positioned close to Microcoleaceae genera, but distant from Microcoleus. Romeriopsis is positioned within the Leptolyngbyaceae and is closely related to Alkalinema. The secondary structures of the D1-D1, Box B, V2 and V3 helices corroborate with the phylogenetic results. Furthermore, our study supports previous observations of polyphyletic Oscillatoriales families and reinforces the need for their taxonomical revision.

microbiology↗

bioRxiv2020

A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion into eukaryotic cells

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454-484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

biophysics↗

bioRxiv2019

The immunomodulatory effect of IrSPI, a tick salivary gland serine protease inhibitor involved in Ixodes ricinus tick feeding

Ticks are strict hematophagous arthropods and are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Moreover, they are second only to mosquitoes as vectors of human pathogens. Hard tick feeding is a slow process--taking up to several days for repletion prior to detachment--and necessitates extended control over the host response. The success of the feeding process depends upon injection of saliva by tick, which not only controls host haemostasis and wound healing, but also subverts the host immune response to avoid tick rejection during this long-lasting process. In turn, the manipulation of the host immune response creates a favourable niche for the survival and propagation of diverse tick-borne pathogens transmitted during feeding. Here, we report on the molecular and biochemical features and functions of IrSPI, an Ixodes ricinus salivary serine protease inhibitor involved in blood meal acquisition. Our results show that IrSPI harbours the typical conformational fold of Kunitz type I serine protease inhibitors and that it functionally inhibits the elastase and, to a lesser extent, chymotrypsin. We also show that IrSPI is injected into the host during feeding. Crucially, we found that IrSPI has no impact on tissue factor pathway-induced coagulation, fibrinolysis, apoptosis, or angiogenesis, but a strong effect on immune cells. IrSPI affects antigen-presenting macrophages by hampering IL-5 production. In addition, IrSPI represses proliferation of mitogen-stimulated CD4+ cells. The inhibition of T cell proliferation was associated with marked reductions in pro-inflammatory cytokine secretion. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in successful I. ricinus tick feeding.\n\nAuthor summaryTicks are the most important vector influencing both human and animal health in Europe, where Ixodes ricinus is the most abundant tick species. Ticks feed on animal or human blood for an extended period, during which their saliva allows both feeding and pathogen transmission by interfering with native host responses. A better understanding of tick-host-pathogen interactions is central to the discovery of improved control methods. Within this context, we previously identified IrSPI as an I. ricinus salivary molecule implicated in both tick feeding and bacterial transmission. This serine protease inhibitor was almost characterised as an elastase inhibitor, and here, we show IrSPI overexpression in several tick organs--especially salivary glands--during blood feeding. We demonstrate that IrSPI is injected into the host through saliva, and despite having no impact on endothelial cell angiogenesis or apoptosis during blood feeding, we report an immunomodulatory role, whereby CD4+ T lymphocyte proliferation is repressed and where the cytokine secretion pattern of both splenocytes and macrophages is modified. Our study provides new insights into the complex armament developed by ticks to overcome the host response, and uncovers a potential vaccine target for disruption of feeding processes and pathogen transmission.

animal behavior and cognition↗