Biology subjects

Lapouge, K.

Publications and source records attributed to Lapouge, K..

11 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

The cancer-promoting enzyme PKM2 binds RNA via a positively charged regulatory patch

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that coordinates energy production with biosynthetic demands in physiologically proliferating and cancer cells. PKM2 has also emerged as a non-canonical RNA-binding protein. Here, we combine targeted PKM2 mutagenesis with RNA-protein interaction assays, biochemical and biophysical analyses to define how RNA binding relates to PKM2 allosteric control and oligomeric state. We show that the allosteric activator fructose-1,6-bisphosphate (FBP) strongly reduces PKM2 binding to RNA. Mutants with impaired oligomerization show reduced RNA binding, and RNA association is favored by the tetrameric state. A positively charged surface patch in the FBP-binding region is essential for RNA binding and displays emergent properties. Single-residue variants further link RNA association to FBP-responsive tetrameric conformations. Our data integrate RNA binding with PKM2 allosteric regulation.

molecular biology↗

bioRxiv2026

The hearing-essential intracellular domain of PCDH15 reveals a new layer of auditory mechanotransduction

Current models of mechanotransduction in inner-ear hair cells explain how force is transmitted through the extracellular tip link but provide little insight into how force is propagated beneath the plasma membrane. Although the CD2 isoform of PCDH15 has been shown to be essential for hearing in mature mammalian hair cells, its structural and mechanical properties have remained largely unknown. Here, we combine computational sequence analysis, orthogonal biophysical characterization and small-angle X-ray scattering (SAXS) to show that the hearing-essential CD2 intracellular domain is an intrinsically disordered region (IDR) that behaves as a highly expanded acidic polyampholyte. CD2 remains predominantly monomeric in solution and occupies a larger conformational space at physiological-like ionic strength, whereas inclusion of the isoform-shared common region increases self-association. CD2 also contains a conserved regulatory interaction platform, providing a potential link between its polymer properties and cellular regulation. Together, these findings identify the CD2 intracellular domain as a polymer with biophysical properties consistent with a mechanically responsive structural element. Furthermore, they provide a framework for investigating how intracellular polymer mechanics may contribute to force transmission and adaptation in auditory mechanotransduction.

biophysics↗

bioRxiv2026

Molecular basis of mitogen-activated protein kinase ERK2 activation by its upstream kinase MEK1

The RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) pathway relays extracellular signals into a cellular response and its dysregulation leads to many pathologies, particularly cancer. Here, we determined cryo-EM structures of the MAP2K MEK1 activating its substrate MAPK ERK2, the final event in the cascade. We define the molecular details of specificity and phosphoryl transfer to the tyrosine of the ERK2 activation loop and examine the mechanism of substrate recognition using solution techniques and molecular dynamics. Binding of the substrate MAPK leads to release of the MAP2K catalytic machinery, a mechanism enhanced in many gain of function disease mutations. Further, we observe that ERK2 release is not required for nucleotide exchange, suggesting how a processive mechanism could proceed. Our data advance the understanding of MAPK signalling and provide a starting point for drug development. One-Sentence SummaryCryo-EM structures of the MEK1-ERK2 complex reveal details of cellular signal transmission.

molecular biology↗

bioRxiv2026

Selective class IIa HDAC inhibition reverses diastolic dysfunction in cardiometabolic HFpEF

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent cardiometabolic syndrome with yet no effective therapies. Here, we report that selectively class IIa histone deacetylases (HDACs) but no other classes of HDACs are enzymatically activated in hearts from HFpEF patients and cardiometabolic HFpEF animal models. Cell type-specific and enzymatic activity-specific genetic loss-of-function models of the cardiomyocyte-enriched class IIa HDAC family member HDAC4 and the pharmacological class IIa HDAC-selective inhibitor TMP195 prevent and reverse diastolic dysfunction and exercise intolerance in cardiometabolic HFpEF in vivo. In contrast to pan-HDAC inhibition no adverse effects are observed. Despite its well-known non-enzymatic role as transcriptional repressor, we found that specifically enzymatic activation of HDAC4 has little direct effects on cardiomyocyte-intrinsic gene expression. Instead, non-epigenetic actions lead to endothelial activation via altered cardiocrine signaling. We discovered that selective enzymatic class IIa HDAC inhibition is a new therapeutic concept to combat cardiac HFpEF.

molecular biology↗

bioRxiv2026

Short linear motifs - Unexplored players driving Toxoplasma gondii infection

Pathogens infect hosts by interacting with host proteins and exploiting their functions to their advantage. Short linear motifs, small functional regions within intrinsically disordered protein regions, are common mediators of host-pathogen protein interactions. While motifs have been more extensively studied in viruses and bacteria, the extent to which eukaryotic unicellular parasites use motifs during infection remains unexplored. Toxoplasma gondii is a widespread intracellular Apicomplexan parasite capable of infecting all warm-blooded animals and invading any of their nucleated cells. Toxoplasmas secreted proteins are key in interacting with host proteins during infection, making them potential sources for motifs. To highlight the role of motifs in Toxoplasma gondii infection, we curated 21 known motif instances in Toxoplasma proteins from the scientific literature. To identify more motifs in Toxoplasma secreted proteins, we developed a computational pipeline that annotates putative motif matches with structural and functional features. Through this approach, we identified a set of 24,291 motif matches in 295 secreted proteins. We highlight strategies for further prioritisation of likely functional motif matches by focusing on integrin motifs, degrons and TRAF6-binding motifs. We subjected four predicted TRAF6-binding motifs to experimental validation, supporting the predicted motifs in the Toxoplasma proteins RON10 and GRA15. Our motif predictions provide a valuable resource for generating hypotheses and designing experiments to study infection mechanisms. The characterisation of motifs in Toxoplasma will be key to understanding the molecular principles underlying its broad host range and more comprehensive Apicomplexan infection strategies. ImportanceToxoplasma gondii is a widely distributed intracellular parasite that achieves a successful infection by interacting with different host cell proteins. Short linear motifs are small functional modules found in unstructured protein regions and recognised by folded protein domains. Given that unstructured protein regions are a common feature of Toxoplasmas proteins, we hypothesise that motifs play important roles during its infection cycle. Here, we highlight the role of motifs during the Toxoplasma host cell invasion cycle through a curated set of motif examples. Through a computational pipeline, we predict thousands of motifs in secreted proteins, outline strategies for working with these predictions and finally experimentally test proteins containing a motif involved in the innate immune response, successfully showing the binding of two motifs. Our work provides a resource for further motif testing in Toxoplasma proteins, aiming at understanding the molecular mechanisms of its infection strategies and its broad host range.

bioinformatics↗

bioRxiv2025

Molecular co-accessibility identifies coordinated regulation between distant cis-regulatory elements

In metazoans, gene expression is typically regulated by a cis-regulatory landscape (CRL) composed of a promoter and multiple enhancers. How these cis-regulatory elements (CREs) coordinate their function across large genomic distances remains unclear. For example, is the simultaneous activation of multiple enhancers required to promote transcription? Here, we combined single-molecule footprinting with long-read sequencing to quantify how often chromatin accessibility and transcription factor binding co-occur across entire CRLs in the Drosophila genome. Analysis of thousands of individual DNA molecules at each locus revealed that CREs form a specific network with shared single-molecule chromatin accessibility profiles. Co-accessibility is not limited to adjacent CREs, and is frequently observed between CREs brought into proximity by chromatin looping. Co-accessible CREs exhibit strong coordination in their cell type-specific accessibility, linking enhancer activity with transcription activation. Our data uncover dependencies between CREs genome-wide, and suggest that coordinated enhancer activation is a widespread mechanism regulating gene expression.

genomics↗

bioRxiv2024

The Drosophila RNA binding protein Hrp48 binds a specificRNA sequence of the msl-2 mRNA 3' UTR to regulatetranslation

Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3 untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3 UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.

biophysics↗

bioRxiv2024

Trim66 paternal deficiency causes intrauterine overgrowth

The tripartite motif-containing protein 66 (TRIM66, also known as TIF1-delta) is a PHD-Bromo containing protein primarily expressed in post-meiotic male germ cells known as spermatids. Biophysical assays showed that TRIM66 PHD-Bromo domain binds to H3 N-terminus only when lysine 4 is unmethylated. We addressed TRIM66s role in reproduction by loss-of-function genetics in the mouse. Males homozygous for Trim66-null mutations produced functional spermatozoa. Round spermatids lacking TRIM66 upregulated a network of genes involved in histone acetylation and H3K4 methylation. Profiling of H3K4me3 patterns in the sperm produced by Trim66-null mutant showed minor alterations below statistical significance. Unexpectedly, Trim66-null males, but not females, sired pups overweight at birth, hence revealing that Trim66 mutations cause a paternal effect phenotype.

developmental biology↗

bioRxiv2022

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless

Unwinding RNA secondary structures by RNA helicases is essential for RNA metabolism. How the basic unwinding reaction of DExH-type helicases is regulated by their accessory domains is unresolved. Here, we combine structural and functional analyses to address this challenge for the prototypic DExH RNA helicase maleless (MLE) from Drosophila. We captured the helicase cycle of MLE with multiple structural snapshots. We discovered that initially, dsRBD2 flexibly samples substrate dsRNA and aligns it with the open helicase tunnel. Subsequently, dsRBD2 releases RNA and associates with the helicase core, leading to closure of the tunnel around ssRNA. Structure-based MLE mutations confirm the functional relevance of the structural model in cells. We propose a molecular model in which the dsRBD2 domain of MLE orchestrates large structural transitions that depend on substrate RNA but are independent of ATP. Our findings reveal the fundamental mechanics of dsRNA unwinding by DExH helicases with high general relevance for dosage compensation and specific implications for MLEs human orthologue DHX9/RHA mechanisms in disease.

molecular biology↗

bioRxiv2022

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex

Folding of nascent chains emerging from the ribosome is a challenge in cellular protein homeostasis, which in eukaryotes is met by an Hsp70 chaperone triad directly binding at the ribosomal tunnel exit. The conserved ribosome-associated complex (RAC) consists of the non-canonical Hsp70 Ssz1 and the J-domain protein Zuotin (Zuo1), which in fungi acts together with the canonical Hsp70 protein Ssb. Here, we determined high-resolution cryo-electron microscopy structures of RAC bound to the 80S ribosome. RAC adopts two distinct conformations accommodating continuous ribosomal rotation by a flexible lever arm. The heterodimer is held together by a tight interaction between the Ssz1 substrate-binding domain (SBD) and the N-terminus of Zuo1, with additional contacts between the Ssz1 nucleotide-binding domain (NBD) and the Zuo1 J- and ZHD domains that form a rigid unit. The Zuo1 HPD-motif conserved in J-proteins is masked by the Ssz1 NBD, different from the canonical Hsp70 J-protein contact, however, allowing to position Ssb for activation by Zuo1. Our data provide the basis for understanding how RAC cooperates with Ssb at the ribosome in dynamic nascent chain interaction and protein folding.

biochemistry↗