Biology subjects

Michaux, C.

Publications and source records attributed to Michaux, C..

10 recordsLinked to original sources

bioRxiv2026

Molecular dynamics insights into biomineralisation mediated by acidic intrinsically disordered proteins: a case study of molluscan Aspein from the pearl oyster Pinctada fucata

Biomineralisation is a ubiquitous phenomenon that still fascinates the scientific community. Mineralised structures are most notably encountered in marine lifeforms the shell or exoskeleton of which are formed by precipitating specific calcium carbonate (CaCO3) polymorphs, known as amorphous (ACC), vaterite, aragonite, and calcite. To control crystalline polymorphism in their shell layers, bivalves have evolved strategies involving ion-binding secretomes composed of shell matrix proteins (SMPs). Secreted in the prismatic layer of the pearl oyster Pinctada fucata, Aspein is an unusually acid-rich protein exclusively aspartic and famously recognised as the most acidic SMP known to date. Through in vitro crystallisation experiments, Aspein was shown to select calcite over aragonite in modern seawater conditions. However, how it exerts its selectivity with respect to its structural properties remains enigmatic. By combining sequence-based predictions with all-atom molecular dynamics simulations, we unveiled that Aspein is an intrinsically disordered organic matrix protein (IDOMP) undergoing ion-specific chain collapse and phase separation. Thanks to its unique Asp density and conformational fuzziness, Aspein first sequesters Ca2+ then attracts CO32- ions before stabilising ACC and calcite-like phases, while precluding the incorporation of Mg2+ to avoid aragonite formation. Indeed, via Asp-Ca2+ bridges, structure-less clusters coalescence into supersaturated protein-CaCO3 assemblies initiates calcite crystallisation. Therefore, Aspein plasticity, condensation susceptibility, and aspartic density critically modulate the pathway that defines CaCO3 polymorphism by inhibiting nucleation and controlling the growth of biogenic minerals. From a broader perspective, our study underpins some of the molecular and physico-chemical principles governing biomineralisation processes mediated by IDOMPs, the overlooked, yet prevalent, role of which was mechanistically unexplored.

biophysics↗

bioRxiv2025

Unveiling the effect of phosphorylation on the structural and aggregation properties of the amyloidogenic intrinsically disordered protein DPF3a

The double plant homeodomain fingers 3a (DPF3 isoform a) is a human epigenetic regulator involved in chromatin remodelling, cell division, and ciliogenesis. Most notably, this protein is deregulated in various cancer types and neurodegenerative diseases. In our previous work, the disorder nature of DPF3a, as well as its propensity to aggregate into amyloid fibrils, have been highlighted, making it an amyloidogenic intrinsically disordered protein (IDP). Due to their high chain accessibility, IDPs structure and function are modulated by phosphorylation. It has been reported that phosphorylation of DPF3a at S348 (pS348) by the casein kinase 2 (CK2) is implicated in cardiac hypertrophy. CK2 can also phosphorylate DPF3a at S138 (pS138), which is also located in an intrinsically disordered region (IDR). However, no structural information is available on phosphorylated DPF3a. In the present study, we investigated the effect of phosphorylation on DPF3a structural and aggregation properties. Two single-mutated phosphomimetics (S138E and S348E) were characterised in vitro and compared to DPF3a WT, while in silico analyses were performed on pS138 and pS348 to assess structural changes at the molecular level. Circular dichroism and fluorescence spectroscopy revealed that both phosphomimetics are hybrid IDPs, with increased turn and antiparallel {beta}-sheet content as well as more buried aromatic residues compared to DPF3a WT, suggesting conformational rearrangements and a more folded N-terminal region. In silico characterisation supported these results, showing that phosphorylation of S138 and S348 induce extended conformation, especially the C-terminal extremity, due to electrostatic repulsion, while local folding occurs due to a proximity with arginine and lysine residues. Furthermore, spectroscopic and microscopic analyses unveiled that S138E and S348E exhibit slower fibrillation kinetics compared to DPF3a WT involving distinct aggregation mechanisms.

biophysics↗

bioRxiv2025

Stress-Induced Iron-Sulfur Cluster Damage as a Conserved Trigger of the Stringent Response

Pathogenic bacteria rely on the stringent response to adapt to the complex and fluctuating conditions encountered within the host. However, the mechanisms by which the stringent response senses host-induced stress remain poorly understood. Here, we identify iron-sulfur (Fe-S) cluster damage as a conserved trigger of the stringent response in major Gram-negative pathogens, including Salmonella enterica, Enterobacter cloacae, and Klebsiella pneumoniae. We demonstrate that Fe-S cluster disruption--caused by oxidative stress or metal imbalance--restricts the intracellular pools of sulfur-containing and branched-chain amino acids, thereby activating the ribosome-associated (p)ppGpp synthetase RelA. Furthermore, we show that iron availability governs recovery from Fe-S cluster damage, modulating the dynamics of the stringent response. Finally, we emphasize the dual role of (p)ppGpp in transcriptional regulation, enhancing bacterial fitness during Fe-S cluster stress while simultaneously promoting virulence by upregulating the SPI-2 type III secretion system. Together, these findings uncover a conserved mechanism by which pathogenic bacteria integrate metabolic stress into adaptive gene regulation and virulence, positioning Fe-S cluster integrity as a pivotal node linking environmental sensing to transcriptional control during infection.

microbiology↗

bioRxiv2025

Atypical features of the disordered N-terminal region of a bacterial outer membrane copper transporter.

Metals like copper (Cu), zinc, and nickel exhibit dual nature, necessitating a tight regulation of their cellular homeostasis to meet physiological demands while preventing toxicity. In bacteria, metal homeostasis involves inner membrane (IM) P-type ATPases and ABC transporters, envelope-spanning tripartite efflux pumps, and outer membrane (OM) pore-forming proteins. Four decades ago, the OM {beta}-barrel protein PcoB was shown to provide an additional layer of Cu resistance in an Escherichia coli strain isolated from the gut of swine fed with Cu supplements. Interestingly, most PcoB homologs contain a poorly conserved disordered N-terminal domain (NTD) rich in histidine (His) and methionine (Met) residues, which are commonly associated with Cu coordination in cuproproteins. This suggests a potential role for the NTD in PcoB-mediated copper efflux. We previously demonstrated that the free-living bacterium Caulobacter vibroides primarily relies on PcoB for Cu homeostasis. Here, we show that the NTD of C. vibroides PcoB is critical for PcoB function and stability, tolerating the swapping with the poorly conserved E. coli PcoB NTD and significant truncations. Unexpectedly, the predicted signal peptide (SP) was dispensable, challenging traditional concepts of protein translocation mechanisms. Moreover, the PcoB NTD plays a surprising role in stabilizing the periplasmic multicopper oxidase PcoA, encoded within the same operon as PcoB, highlighting a new role for an intrinsically disordered region (IDR). ImportanceBacterial copper (Cu) homeostasis is essential for survival in fluctuating environments, yet the role of single outer membrane proteins in this process remains poorly characterized. Our study reveals that the intrinsically disordered N-terminal region of the {beta}-barrel protein PcoB in Caulobacter vibroides plays a critical role in Cu efflux and PcoB stability. Remarkably, this region harbors an atypical signal peptide and contributes to the stability of the periplasmic multicopper oxidase PcoA, suggesting a novel regulatory function for an intrinsically disordered region. These findings challenge existing paradigms of protein targeting and homeostasis, with broad implications for understanding bacterial adaptation to stress.

molecular biology↗

bioRxiv2025

Exploration of the influence of environmental changes on the conformational and amyloidogenic landscapes of the zinc finger protein DPF3a by combining biophysical and molecular dynamics approaches

In the past few years, the double PHD fingers 3 (DPF3) protein isoforms (DPF3b and DPF3a) have been identified as new amyloidogenic intrinsically disordered proteins (IDPs). Although such discovery is coherent and promising in the light of their involvement in proteinopathies, their amyloidogenic pathway remains largely unexplored. As environmental variations in pH and ionic strength are relevant to DPF3 pathophysiological landscape, we therefore enquired the effect of these physicochemical parameters on the protein structural and prone-to-aggregation properties, by focusing on the more disordered DPF3a isoform. In the present study, we exploited in vitro and in silico strategies by combining spectroscopy, microscopy, and all-atom molecular dynamics methods. Very good consistency and complementary information were found between the experiments and the simulations. Acidification unequivocally abrogated DPF3a fibrillation upon maintaining the protein in highly hydrated and expanded conformers due to extensive repulsion between positively charged regions. In contrast, alkaline pH delayed the aggregation process due to loss in intramolecular contacts and chain decompaction, the extent of which was partly reduced thanks to the compensation of negative charge by arginine side chains. Through screening attractive electrostatic interactions, high ionic strength conditions (300 and 500 mM NaCl) shifted the conformational ensemble towards more swollen, heterogeneous, and less H-bonded structures, which were responsible for slowing down the conversion into {beta}-sheeted species and restricting the fibril elongation. For defining the self-assembly pathway of DPF3a, we unveiled that the protein amyloidogenicity intimately communicates with its conformational landscape, which is particularly sensitive to modification of its physicochemical environment. As such, understanding how to modulate DPF3a conformational ensemble will help designing novel protein-specific strategies for targeting neurodegeneration. HighlightsO_LIDPF3a is a polyampholyte IDP, structurally sensitive to environmental changes. C_LIO_LIDPF3a amyloid pathway and propensity can be modulated by pH and ionic strength. C_LIO_LIAcidic condition inhibits fibrillation and maintains DPF3a in an extended state. C_LIO_LIAlkaline pH and ionic strength delay fibrillation by reducing structure collapse. C_LIO_LIDPF3a fibrils exhibit condition-dependent optical-morphological properties. C_LI

biophysics↗

bioRxiv2024

LolA and LolB are conserved in Bacteroidetes and are crucial for gliding motility and Type IX secretion.

In Gram-negative bacteria, lipoproteins are major components of the outer membrane (OM) where they play a variety of roles, from the involvement in membrane biogenesis to virulence. Bacteroidetes, a widespread phylum of Gram-negative bacteria, including free-living organisms, commensals and pathogens, encode an exceptionally high number of outer membrane lipoproteins. These proteins are crucial in this phylum mainly because they are key components of SUS-like nutrient acquisition systems as well as of the Type 9 secretion (T9SS) and gliding motility machineries. The transport of lipoproteins to the OM has mainly been studied in E. coli and relies on the Lol system, composed of the inner membrane extraction machinery LolCDE, the periplasmic carrier LolA and the OM lipoprotein LolB. While most Lol proteins are essential and conserved across Gram-negative bacteria, to date, no LolB homologs have been identified outside of {gamma}- and {beta}-proteobacteria. How lipoproteins reach and are inserted in the OM of Bacteroidetes is not known. Here we identified LolB homologs in Bacteroidetes and disclosed the co-existence of several LolA and LolB in several species. We provide evidence that one LolA (LolA1) and one LolB (LolB1) of F. johnsoniae are devoted to targeting gliding and T9SS lipoproteins to the OM. A proteomic analysis of the OM composition of the lolA1 and lolB1 mutants supports this evidence. Furthermore, we show that, while LolB1 and LolA1 have conserved functions in Bacteroidetes, they are functionally different from their E. coli counterparts. We also show that surface lipoprotein transport is LolA and LolB independent. Finally, the finding that, in the absence of LolA and LolB homologs, lipoproteins still localize to the OM, suggests the presence in Bacteroidetes of yet unidentified LolAB-alternative lipoprotein transport pathways. In conclusion, Bacteroidetes have evolved different and more complex lipoprotein transport pathways than other Gram-negative bacteria and further research is required to uncover their complexity. SignificanceIn Gram-negative bacteria, lipoproteins are key components of the outer membrane (OM), essential for functions like membrane biogenesis and virulence. Bacteroidetes, a widespread phylum, encode a high number of OM lipoproteins crucial for nutrient acquisition, Type IX secretion, and gliding motility. While lipoprotein transport in E. coli depends on the Lol system, LolB homologs were previously unidentified outside {gamma}- and {beta}-proteobacteria. Here we identify LolB homologs in Bacteroidetes, revealing the co-existence of multiple LolA and LolB proteins in various species. In F. johnsoniae, LolA1 and LolB1 specifically target gliding and Type 9 secretion system lipoproteins to the OM. Despite this, lipoproteins still localize to the OM without LolA and LolB, suggesting alternative transport pathways. These findings indicate that Bacteroidetes have evolved more complex lipoprotein transport mechanisms than other Gram-negative bacteria, requiring further research to fully understand them.

microbiology↗

bioRxiv2024

Intrinsic Disorder and Salt-dependent Conformational Changes of the N-terminal TFIP11 Splicing Factor

Tuftelin Interacting Protein 11 (TFIP11) was recently identified as a critical human spliceosome assembly regulator, interacting with multiple spliceosome proteins and localises in several membrane-less organelles. However, there is a lack of structural information on TFIP11, limiting the rationalisation of its biological role. TFIP11 has been predicted as a highly disordered protein, and more specifically concerning its N-terminal (N-TER) region. Intrinsically disordered proteins (IDPs) lack a defined tertiary structure, existing as a dynamic conformational ensemble, favouring their role as hubs in protein-protein and protein-RNA interaction networks. Furthermore, IDPs are involved in liquid-liquid phase separation (LLPS), driving the formation of subnuclear compartments. Combining disorder prediction, molecular dynamics, and spectroscopy methods, this contribution shows the first evidence TFIP11 N-TER may be described as a polyampholytic IDP, exhibiting a structural duality with the coexistence of ordered and disordered assemblies, depending on the ionic strength of the protein environment. Increasing the salt concentration enhances the protein conformational flexibility, presenting a fuzzier conformational landscape, a more globule-like shape, and an unstructured arrangement that could favour LLPS segregation and protein-RNA interaction. The regions mostly composed of charged and hydrophilic residues are the most impacted, including the G-Patch domain which is of crucial importance to TFIP11 function.

bioinformatics↗

bioRxiv2023

Improved RNA stability estimation through Bayesian modeling reveals most bacterial transcripts have sub-minute half-lives

RNA decay is a crucial mechanism for regulating gene expression in response to environmental stresses. In bacteria, RNA-binding proteins (RBPs) are known to be involved in post-transcriptional regulation, but their global impact on RNA half-lives has not been extensively studied. To shed light on the role of the major RBPs ProQ and CspC/E in maintaining RNA stability, we performed RNA sequencing of Salmonella enterica over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq) in the presence and absence of these RBPs. We developed a hierarchical Bayesian model that corrects for confounding factors in rifampicin RNA stability assays and enables us to identify differentially decaying transcripts transcriptome-wide. Our analysis revealed that the median RNA half-life in Salmonella in early stationary phase is less than 1 minute, a third of previous estimates. We found that over half of the 500 most long-lived transcripts are bound by at least one major RBP, suggesting a general role for RBPs in shaping the transcriptome. Integrating differential stability estimates with CLIP-seq revealed that approximately 30% of transcripts with ProQ binding sites and more than 40% with CspC/E binding sites in coding or 3 untranslated regions decay differentially in the absence of the respective RBP. Analysis of differentially destabilized transcripts identified a role for ProQ in the oxidative stress response. Our findings provide new insights into post-transcriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression. Significance StatementTogether with transcription and translation, RNA decay is one of the major processes governing protein production. Here, we have developed a new statistical approach that corrects for confounding effects when estimating RNA decay rates from RNA-seq in bacteria. Our more accurate decay rate estimates indicate that Salmonella transcripts have half-lives about three times shorter than previously thought. This approach allowed us to measure the effects of RNA-binding proteins (RBPs) on decay rates, identifying large cohorts of transcripts with changes in stability following RBP deletion and conditions where post-transcriptional regulation affects survival. Our method should lead to a reevaluation of RNA stability estimates across diverse bacteria and new insights into the role of RBPs in shaping the transcriptome.

microbiology↗

bioRxiv2022

Grad-seq analysis of Enterococcus faecalis and Enterococcus faecium provides a global view of RNA and protein complexes in these two opportunistic pathogens

Enterococcus faecalis and Enterococcus faecium are major nosocomial pathogens. Despite their relevance to public health and their role in the development of bacterial antibiotic resistance, relatively little is known about gene regulation in these species. RNA-protein complexes serve crucial functions in all cellular processes associated with gene expression, including post-transcriptional control mediated by small regulatory RNAs (sRNAs). Here, we present a new resource for the study of enterococcal RNA biology, employing the Grad-seq technique to comprehensively predict complexes formed by RNA and proteins in E. faecalis V583 and E. faecium AUS0004. Analysis of the generated global RNA and protein sedimentation profiles led to the identification of RNA-protein complexes and putative novel sRNAs. Validating our data sets, we observe well-established cellular RNA-protein complexes such as the 6S RNA-RNA polymerase complex, suggesting that 6S RNA-mediated global control of transcription is conserved in enterococci. Focusing on the largely uncharacterized RNA-binding protein KhpB, we use the RIP-seq technique to predict that KhpB interacts with sRNAs, tRNAs, and untranslated regions of mRNAs, and might be involved in the processing of specific tRNAs. Collectively, these datasets provide departure points for in-depth studies of the cellular interactome of enterococci that should facilitate functional discovery in these and related Gram-positive species. Our data are available to the community through a user-friendly Grad-seq browser that allows interactive searches of the sedimentation profiles (https://resources.helmholtz-hiri.de/gradseqef/).

microbiology↗

bioRxiv2022

Reactive oxygen species-based sensing by a single chemoreceptor controls bacterial chemotaxis to copper

Chemotaxis is a widespread strategy used by unicellular and multicellular living organisms to maintain their fitness in stressful environments. We previously showed that bacteria can trigger a negative chemotactic response to a copper (Cu)-rich environment. Cu ions toxicity on bacterial cell physiology has been mainly linked to mismetallation events and ROS production, although the precise role of Cu-generated ROS remains largely debated. Here, we found that the cytoplasmic Cu ions content mirrors variations of the extracellular Cu ions concentration and triggers a dose-dependent oxidative stress, which can be abrogated by superoxide dismutase and catalase overexpression. The inhibition of ROS production in the cytoplasm not only improves bacterial growth but also impedes Cu-chemotaxis, indicating that ROS derived from cytoplasmic Cu ions mediate the control of bacterial chemotaxis to Cu. We also identified the Cu chemoreceptor McpR, which binds Cu ions with low affinity, suggesting a labile interaction. In addition, we demonstrate that the cysteine 75 and histidine 99 within the McpR sensor domain are key residues in Cu chemotaxis and Cu coordination. Finally, we discovered that in vitro both Cu(I) and Cu(II) ions modulate McpR conformation in a distinct manner. Overall, our study provides mechanistic insights on a redox-based control of Cu chemotaxis, indicating that the cellular redox status can play a key role in bacterial chemotaxis.

microbiology↗