Biology subjects

Hamelin, R.

Publications and source records attributed to Hamelin, R..

12 recordsLinked to original sources

bioRxiv2026

A Robust and Generalizable Low-Input Spatial Proteomics Workflow Enabling Deep Proteome Coverage

Spatial proteomics has rapidly emerged as a powerful approach for deciphering the molecular architecture of complex tissues, offering critical insights into how local proteome composition shapes tissue function. In this work, we aimed to broaden access to mass spectrometry-based spatial proteomics by putting in place a pipeline relying exclusively on instrumentation commonly available in standard histology, imaging, and proteomics facilities. The resulting workflow integrates high-resolution whole-slide imaging, image-based region selection, streamlined low-input sample processing, and a high-sensitivity method optimized on the Orbitrap Exploris platform. To demonstrate the potential of this approach, we applied it to the choroid plexus (ChP), a highly specialized but understudied brain structure whose spatial molecular organization remains poorly characterized. Analysis of ChP samples from three brain compartments across mice of different age and sex quantified more than 8,000 protein groups across the dataset from minimal input material, providing unprecedented proteome-wide resolution of ChP spatial heterogeneity. Our findings establish a foundational spatial proteomic atlas of the ChP and highlight new opportunities for investigating its roles in aging, neurological disease, and central nervous system infection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=133 HEIGHT=200 SRC="FIGDIR/small/738934v2_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@1b6020eorg.highwire.dtl.DTLVardef@40fd9aorg.highwire.dtl.DTLVardef@c9b5b3org.highwire.dtl.DTLVardef@b81e0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

FIKK1, a member of the FIKK kinase family, phosphorylates VAR2CSA and regulates adhesion of Plasmodium falciparum-infected erythrocytes to the placental receptor CSA

Plasmodium falciparum promotes the adhesion of infected erythrocytes (IEs) to host cells by extensively remodeling their surface. For this process, the parasite exports a large number of proteins to its host erythrocyte, including members of the P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1) adhesin family and members of the FIKK family. Several FIKK have been shown to play a role in P. falciparum virulence, notably affecting IES cell surface remodeling, rigidity and cytoadhesion. VAR2CSA, a member of the PfEMP1 adhesin family, is associated with IES sequestration in the placenta and has been shown to be phosphorylated. In view of the previously described importance of VAR2CSA phosphorylation, we investigated the role of FIKK1. We show that FIKK1 is capable of phosphorylating VAR2CSA in vitro, and that this phosphorylation increases the binding of recombinant VAR2CSA to the placental receptor chondroitin sulphate A (CSA). In an inducible transgenic cell line expressing HA-tagged FIKK1, immunofluorescence assays indicate that the kinase localizes to punctuated foci within Maurers Cleft, similarly to VAR2CSA. Rapamycin-induced knock out of FIKK1 reduces IEs cytoadhesion to CSA, even though levels of VAR2CSA are not affected. In vitro phosphorylation assays show that FIKK1 can phosphorylate recombinant DBL1-3 domains on several residues, including S429 and T934, previously implicated in in vitro binding and IES cytoadhesion to CSA. Taken together, these data support a model whereby FIKK1 contributes to placental malaria virulence through IEs sequestration mediated by VAR2CSA phosphorylation. Having no orthologs in mammals, this orphan kinase therefore represents an attractive target for the development of drugs against placental malaria. Author summarySequestration of Plasmodium falciparum infected erythrocytes (IEs) in the placenta is a hallmark of placental malaria and a major driver of adverse maternofetal outcomes. This process is mediated by VAR2CSA, a member of the P. falciparum Erythrocyte Membrane Protein (PfEMP1) family. VAR2CSA binds to chondroitin sulfate A (CSA) on the placental syncytium, facilitating IEs sequestration. In a previous study, we demonstrated that endogenous VAR2CSA is phosphorylated and identified specific phosphosites important for its cytoadhesive function. To elucidate the molecular mechanisms underlying these post-translational modifications, we examined the role of the P. falciparum FIKK1 kinase in VAR2CSA phosphorylation and its impact on IEs adhesion. Using a FIKK1::HA conditional knockout transgenic line, we found that FIKK1 deletion impairs IEs cytoadhesion, likely due to altered VAR2CSA phosphorylation. Importantly, both endogenous and recombinant FIKK1 interact with and phosphorylate the extracellular region of VAR2CSA. Furthermore, recombinant FIKK1 also enhances VAR2CSA binding to CSA in vitro and phosphorylates a residue previously identified as important for CSA binding and IEs adhesion. Collectively, these findings highlight a pivotal role for FIKK1 in placental adhesion and underscore the potential of targeting this kinase family for interventions against placental malaria.

biochemistry↗

bioRxiv2025

Tissue-specific restriction of TE-derived regulatory elements safeguards cell-type identity

Transposable elements (TEs) have extensively reshaped the cis-regulatory landscape of mammalian genomes, yet the mechanisms that govern their context-specific activity remain incompletely understood. KRAB zinc finger proteins (KZFPs), a large family of transcription factors specialized in TE recognition, are known repressors of TE-derived regulatory activity through TRIM28-mediated H3K9me3 deposition. Here, we expand this paradigm by uncovering noncanonical relationships between TEs and KZFPs. By generating a comprehensive epigenomic map of KZFP-bound TEs, we find that the regulatory activity of ancient mammalian L2/MIR elements is broadly delineated by KZFP binding patterns, despite low H3K9me3 enrichment. We further dissect this relationship by investigating the function of ZNF436, a non-canonical KZFP, highly expressed during in vivo human fetal heart development. Using loss-of-function approaches, we show that ZNF436 preserves cardiomyocyte function by promoting cardiac gene expression while restricting the activation of alternative lineage programs. Mechanistically, ZNF436 recruits specialized SWI/SNF chromatin remodeling complexes to limit the accessibility of L2/MIR-derived enhancers, many of which are active in non-cardiac tissues. These findings reveal a noncanonical, TRIM28-independent role for KZFPs in shaping cell-type-specific regulatory landscapes and emphasize the importance of repressing alternative regulatory programs alongside activating lineage-specific ones to safeguard cell identity.

genomics↗

bioRxiv2024

An updated proteomic analysis of Drosophila hemolymph after bacterial infection

Using an in-depth Mass Spectrometry based proteomics approach, we provide a comprehensivecharacterization of the hemolymphatic proteome of adult flies upon bacterial infection. We detected and quantified changes in abundance of several known immune regulators and effectors, including multiple antimicrobial peptides, peptidoglycan-binding proteins and serine proteases. Comparison to previously published transcriptomic analyses reveals a partial overlap with our dataset, indicating that many proteins released into the hemolymph upon infection may not be regulated at the transcript level. Among them, we identify a set of muscle-derived proteins released into the hemolymph upon infection. Finally, our analysis reveals that infection induces major changes in the abundance of proteins associated with mitochondrial respiration. This study uncovers a large number of previously undescribed proteins potentially involved in the immune response.

immunology↗

bioRxiv2024

Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

Alpha-synuclein (aSyn) post-translational modifications (PTMs), particularly phosphorylation at serine 129 and C-terminal truncations, are highly enriched in Lewy bodies (LBs), Lewy neurites, and other types of aSyn pathological aggregates in the brain of patients with Parkinsons disease (PD) and other synucleinopathies. However, our knowledge about the precise role of PTMs in regulating the different stages of pathology formation, neurodegeneration, and aSyn pathology spreading remains incomplete. In this work, we applied a systematic approach to address this knowledge gap with an emphasis on mapping and elucidating the role of post-fibrillization C-terminal aSyn truncations in regulating the uptake, processing, seeding activity, and formation of LB-like inclusions and maturations in a well-established neuronal seeding model that recapitulates all the stages leading to LB formation and neurodegeneration. Our work shows that C-terminal cleavage of aSyn fibrils at multiple sites is a conserved process that occurs rapidly after and during the formation of intracellular LB-like aSyn inclusions in all neuronal seeding models. Interestingly, blocking the cleavage of internalized fibrils does not influence their seeding activity, whereas inhibiting the enzymes that regulate the cleavage of newly formed fibrils (e.g., calpains 1 and 2) significantly alters the formation of LB-like inclusions. We also show that C-terminal truncations, in combination with other PTMs, play a crucial role in regulating the interactome and remodeling of newly formed aSyn fibrils, including their shortening, lateral association, and packing during LB formation and maturation. Altogether, our results demonstrate that post-fibrillization C-terminal truncations have a differential role at different stages of aSyn aggregation and pathology formation. These insights, combined with the abundance of truncated aSyn species in LBs, have significant implications in understanding aSyn pathological diversity and developing therapeutic strategies targeting the C-terminus of aSyn or its proteolytic processing.

neuroscience↗

bioRxiv2024

Organ-specific electrophile responsivity mappingin live C. elegans

Proximity labeling technologies are limited to indexing localized protein residents. Such data-- although valuable--cannot inform on small-molecule responsivity of local residents. We here bridge this gap by demonstrating in live C. elegans how electrophile-sensing propensity in specific organs can be quantitatively mapped and ranked. Using this method, >70% of tissue-specific responders exhibit novel responsivity, independent of tissue-specific abundance. One responder, cyp-33e1--for which both human and worm orthologs are electrophile responsive--marshals stress-dependent gut functions, despite manifesting uniform abundance across all tissues studied. Cyp-33e1s localized electrophile responsivity operates site-specifically, triggering multifaceted responses: electrophile sensing through the catalytic-site cysteine results in partitioning between enzyme inhibition and localized production of a critical metabolite that governs global lipid availability, whereas a rapid dual-cysteine site-specific sensing modulates gut homeostasis. Beyond pinpointing chemical actionability within local proteomes, tissue-specific electrophile responsivity mapping illuminates otherwise intractable locale-specific metabolite signaling and stress response programs influencing organ-specific decision-making. IN BRIEFContext-specific protein reactivity is a cornerstone of biological stress responsivity, and thus has important ramifications for drug discovery; no current method can inform on these parameters. Here we debut a method that cartographs locale-specific actionability to reactive metabolites; Cyp-33e1, a gut-specific responder, emerged to partition between electrophile-driven localized enzymatic turnover that triggers gut-specific metabolite production shaping global lipid storage, and localized electrophile sensing that marshals global stress response. HIGHLIGHTSO_LILocalis-REX maps organ-specific electrophile-responsive proteins in worms C_LIO_LIHits are neither identified by organ-specific Ultra-ID nor biased by localized expression C_LIO_LIHits are enriched in proteins functionally relevant to stress-related phenotypes C_LIO_LILocalized responsivity of gut-specific hit, cyp-33e1, shapes global lipid availability C_LI

molecular biology↗

bioRxiv2024

Casein Kinases 2-dependent phosphorylation of the placental ligand VAR2CSA regulates Plasmodium falciparum-infected erythrocytes cytoadhesion

Placental malaria is characterized by the massive accumulation and sequestration of infected erythrocytes in the placental intervillous blood spaces, causing severe birth outcomes. The variant surface antigen VAR2CSA is associated with Plasmodium falciparum sequestration in the placenta via its capacity to adhere to chondroitin sulfate A. We have previously shown that the extracellular region of VAR2CSA is phosphorylated on several residues and that the phosphorylation enhances the adhesive properties of CSA-binding infected erythrocytes. Here, we aimed to identify the kinases mediating this phosphorylation. Here, we report that human and Plasmodium falciparum Casein Kinase 2 are involved in the phosphorylation of the extracellular region of VAR2CSA. We notably show that both CK2 can phosphorylate the extracellular region of recombinant and immunoprecipitated VAR2CSA. Mass spectrometry analysis of recombinant VAR2CSA phosphorylated by recombinant Human and P. falciparum CK2 combined with site-directed mutagenesis led to the identification of residue S1068 in VAR2CSA, which is phosphorylated by both enzymes and is associated with CSA binding. Furthermore, using CRISPR/Cas9 we generated a parasite line in which phosphoresidue, S1068, was changed to alanine. This mutation strongly impairs infected erythrocytes adhesion by abolishing VAR2CSA translocation to the surface of infected erythrocytes. We also report that two specific CK2 inhibitors reduce infected erythrocytes adhesion to CSA and decrease the phosphorylation of the recombinant extracellular region of VAR2CSA using either infected erythrocytes lysates as a source of kinases or recombinant Human and P. falciparum casein kinase 2. Taken together, these results undoubtedly demonstrate that host and P. falciparum CK2 phosphorylate the extracellular region of VAR2CSA and that this post-translational modification is important for VAR2CSA trafficking and for infected erythrocytes adhesion to CSA.

microbiology↗

bioRxiv2022

Resistance mechanism to Notch inhibition and combination therapy in human T cell acute lymphoblastic leukemia

Gain-of-function mutations in NOTCH1 are among the most frequent genetic alterations in T cell acute lymphoblastic leukemia (T-ALL), making the Notch signaling pathway a promising therapeutic target for personalized medicine. Yet, a major limitation for long-term success of targeted therapy is relapse due to tumor heterogeneity or acquired resistance. Thus, we performed a genome-wide CRISPR-Cas9 screen to identify prospective resistance mechanisms to pharmacological NOTCH inhibitors and novel targeted combination therapies to efficiently combat T-ALL. Mutational loss of Phosphoinositide-3-Kinase regulatory subunit 1 (PIK3R1) causes resistance to Notch inhibition. PIK3R1 deficiency leads to increased PIK3/Akt signaling which regulates the cell cycle and spliceosome machinery, both at the transcriptional and post-translational level. Moreover, several therapeutic combinations have been identified, where simultaneous targeting of the cyclin-dependent kinases 4 and 6 (CDK4/6) and NOTCH proved to be the most efficacious in T-ALL xenotransplantation models. Key pointsO_LIMutational loss of PIK3R1 induces resistance to NOTCH1 inhibition in T-ALL C_LIO_LIPharmacological Notch inhibition synergizes with CDK4/6 inhibitors in T-ALL C_LI

cancer biology↗

bioRxiv2021

Regulation of Src tumor activity by its N-terminal intrinsically disordered region

The membrane anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Srcs oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

cancer biology↗

bioRxiv2021

Small Intestinal Resident Eosinophils Maintain Gut Homeostasis Following Microbial Colonisation

Intestinal homeostasis following postnatal microbial colonization requires the coordination of multiple processes including the activation of immune cells, cell-cell communication, the controlled deposition of extracellular matrix, and epithelial cell turnover and differentiation. The intestine harbors the largest frequency of resident eosinophils of all homeostatic organs, yet the functional significance of eosinophil recruitment to this organ has long remained enigmatic. Eosinophils are equipped to both respond to, and modify, their local tissue environment and thus are able to regulate the adaption of tissues to environmental changes. We report a critical role for eosinophils in regulating villous structure, barrier integrity and motility in the small intestine. Notably, the microbiota was identified as a key driver of small intestinal eosinophil activation and function. Collectively our findings demonstrate a critical role for eosinophils in facilitating mutualistic interactions between host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine. HIGHLIGHTSO_LIThe microbiota is a critical regulator of eosinophil activation and turnover C_LIO_LIEosinophils uphold intestinal barrier integrity following microbial colonization C_LIO_LILoss of eosinophils at steady-state results in increased villous blunting and altered intestinal motility C_LI

immunology↗

bioRxiv2019

Variable Genome Evolution in Fungi After Transposon-Mediated Amplification of a Housekeeping Gene

BackgroundTransposable elements (TEs) can be key drivers of evolution, but the mechanisms and scope of how they impact gene and genome function are largely unknown. Previous analyses revealed that TE-mediated gene amplifications can have variable effects on fungal genomes, from inactivation of function to production of multiple active copies. For example, a DNA methyltransferase gene in the wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola) was amplified to tens of copies, all of which were inactivated by Repeat-Induced Point mutation (RIP) including the original, resulting in loss of cytosine methylation. In another wheat pathogen, Pyrenophora tritici-repentis, a histone H3 gene was amplified to tens of copies with little evidence of RIP, leading to many potentially active copies. To further test the effects of transposon-aided gene amplifications on genome evolution and architecture, the repetitive fraction of the significantly expanded Pseudocercospora fijiensis genome was analyzed in greater detail. ResultsThese analyses identified a housekeeping gene, histone H3, which was captured and amplified to hundreds of copies by a hAT DNA transposon, all of which were inactivated by RIP, except for the original. In P. fijiensis the original H3 gene probably was not protected from RIP, but most likely was maintained intact due to strong purifying selection. Comparative analyses revealed that a similar event occurred in five additional genomes representing the fungal genera Cercospora, Pseudocercospora and Sphaerulina. ConclusionsThese results indicate that the interplay of TEs and RIP can result in different and unpredictable fates of amplified genes, with variable effects on gene and genome evolution.

genomics↗

bioRxiv2018

The making of a Lewy body: the role of alpha-synuclein post-fibrillization modifications in regulating the formation and the maturation of pathological inclusions.

Although converging evidence point to alpha-synuclein (a-syn) aggregation and Lewy body (LB) formation as central events in Parkinson's disease (PD), the molecular mechanisms that regulate these processes and their role in disease pathogenesis remain poorly understood. Herein, we applied an integrative biochemical, structural and imaging approach to elucidate the sequence, molecular and cellular mechanisms that regulate LB formation in primary neurons. Our results establish that post-fibrillization C-terminal truncation mediated by calpains 1 and 2 and potentially other enzymes, plays critical roles in regulating a-syn seeding, fibrillization and orchestrates many of the events associated with LB formation and maturation. These findings combined with the abundance of a-syn truncated species in LBs and pathological a-syn aggregates have significant implications for ongoing efforts to develop therapeutic strategies based on targeting the C-terminus of a-syn or proteolytic processing of this region.

neuroscience↗