Biology subjects

Simon, G.

Publications and source records attributed to Simon, G..

17 recordsLinked to original sources

bioRxiv2026

Allosteric covalent inhibition of cereblon through cryptic cysteine targeting

Thalidomide-derived cereblon (CRBN) modulators are among the most clinically impactful targeted protein degradation therapeutics, redirecting the CRL4-CRBN E3 ubiquitin ligase to degrade disease-driving neosubstrates. All current CRBN-targeting IMiDs and CELMoDs engage a single orthosteric pocket, leaving CRBN's conformational landscape largely unexploited pharmacologically. Using cysteine-directed chemoproteomic screening, we identified covalent ligands that engage Cys287, a cryptic allosteric pocket distal to the orthosteric site. Our lead compound, VVD-105917, acts as a pan-inhibitor of IMiD/CELMoD-driven neosubstrate degradation. Cryo-EM structures reveal that covalent Cys287 engagement traps CRBN in its open, inactive conformation, resolving a previously unobserved N-terminal segment and sensor loop. Sequential incubation experiments show the conformational outcome depends on ligand addition order, demonstrating that even potent CELMoD-driven closure can be overridden by allosteric engagement. A recently reported ligand at this same pocket produces the opposite effect, promoting rather than blocking closure, indicating the Cys287 site is mechanistically sign-switchable. These findings establish covalent chemoproteomics as a strategy for discovering and controlling E3 ligase conformational switches.

biochemistry↗

bioRxiv2026

Genetic diversity and phylogeography of swine influenza A virus in 2013-2022 in Europe using a harmonized genotyping nomenclature

Swine Influenza A virus (swIAV) poses constant health threat for both humans and animals. This study comprehensively analysed the genetic diversity and evolution of European swIAVs from 2013 to 2022. To address existing classification gaps, an easily accessible full-genome genotyping tool based on Nextclade was developed alongside a global nomenclature system. Application of this tool to European sequence data revealed unprecedented genetic diversity, successfully identifying 150 swIAV genotypes mixing segments of both swine and human origin. Bayesian phylogeographic models, combined with economic data, identified that the trade of live weaner pigs for fattening drove the transboundary spread of swIAVs across Europe. The study detected repeated human-to-swine reverse-zoonotic events across multiple European countries. These findings clearly highlight the role of commercial pig movements in viral dissemination and emphasize the critical importance of harmonized genomic surveillance to monitor swIAV viral evolution to inform global pre-pandemic preparedness strategies.

microbiology↗

bioRxiv2025

Cysteine reactivity profiling identifies host regulators of Mycobacterium tuberculosis replication in human macrophages

Innate immune cells such as monocytes and macrophages provide the earliest defense against infection by intracellular pathogens by initiating signaling pathways and restricting pathogen replication. However, the full complement of proteins that mediate cell-autonomous immunity remains incompletely defined. Here, we applied cysteine-directed activity-based protein profiling (ABPP) to map proteome-wide cysteine reactivity changes in THP-1 monocytes and primary human monocyte-derived macrophages during Mycobacterium tuberculosis (Mtb) infection. Across both cell types, we quantified 148 cysteine residues with altered reactivity. Genetic perturbation of a subset of proteins harboring these changes significantly impacted Mtb replication, revealing functional links between site-specific cysteine reactivity and antimicrobial defense. These data define previously unrecognized host protein changes during Mtb infection and provide a resource for investigating post-translational events that regulate innate immune responses to intracellular bacteria.

immunology↗

bioRxiv2024

Comparative impact of porcine reproductive and respiratory virus and swine influenza A virus infections on respiratory lymph nodes B cells and macrophages.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) strongly impacts the pig rearing sector due to its persistence in infected animals. Interestingly, although the PRRSV family exhibits considerable genome variability, with the PRRSV-1 and PRRSV-2 subtypes having been finally classified in two different species (Betaarterivirus suid 1 and 2). Both viruses, as well as their derived-attenuated vaccine strains, persist for months, due in part to their ability to delay the appearance of neutralizing antibodies. Thanks to extensive efforts over the past years, we have developed the capability to perform in-depth analysis of the previously poorly understood porcine inverted lymph node (LN). In this study, by comparing the early stages of LN B cell maturation upon PRRSV-1 infection to those induced upon the acute swine influenza A virus infection, we highlighted PRRSV-specific mechanisms, including the expression of PD-L1 in efferent macrophages, the induction of extrafollicular plasmocytes, and the influx of inflammatory monocytes/macrophages. Studies on PRRSV-2 infections report observations compatible with our results, that thus might be generalized to all PRRSV-strains. Moreover, these mechanisms can be compared with those used by the human immunodeficiency virus (HIV) and the murine chronic lymphocytic choriomeningitis virus (LCMV) to hijack the immune response. These similarities can be harnessed to develop new strategies to improve the development of more efficient anti-PRRSV vaccines.

immunology↗

bioRxiv2024

BMP signaling modulations control primitive streak patterning

The primitive streak (PS) is the embryonic structure from which mesoderm and definitive endoderm cells emerge to form organs and fetal tissues. The identities of the cells leaving the PS depend on both the time and the position at which they exit. Three signaling molecules, BMP4, WNT3 and NODAL, play key roles in determining these identities, but it has been difficult to disentangle the contribution of each to this process in the mouse embryo. Using 2D gastruloids that recapitulate early aspects of mouse gastrulation in vitro, we have previously shown that WNT3 and NODAL promote distal PS fates on their own, and proximal PS fates in the presence of BMP4. We now detail further the impact of BMP4 on the cell identities produced in gastruloids. Single cell RNA-seq analysis shows that prolonged exposure to BMP4 results in the presence of all the cell types, including primordial germ cells, which normally form from the most proximal part of the pluripotent epiblast. We then show that reducing the duration or intensity of gastruloid exposure to BMP4 can allow the emergence of more distal cell identities, such as paraxial mesoderm, alongside anterior PS derivatives. These results, which are consistent with embryological studies, provide key elements for understanding how the varied output of the PS is controlled.

developmental biology↗

bioRxiv2024

Major change in swine influenza virus diversity in France owing to emergence and widespread dissemination of a newly introduced H1N2 1C genotype in 2020

Swine influenza A viruses (swIAV) are a major cause of respiratory disease in pigs worldwide, presenting significant economic and health risks. These viruses can reassort, creating new strains with varying pathogenicity and cross-species transmissibility. This study aimed to monitor the genetic and antigenic evolution of swIAV in France from 2019 to 2022. Molecular subtyping revealed a marked increase in H1avN2 cases from 2020 onwards, altering the previously stable subtypes distribution. Whole-genome sequencing and phylogenetic analyses of H1av (1C) strains identified ten circulating genotypes, including five new genotypes, marked by a significant predominance of the H1avN2#E genotype. It was characterized by an HA-1C.2.4, an N2-Gent/84, and internal protein-encoding genes belonging to a newly defined genogroup within the Eurasian avian-like (EA) lineage, the EA-DK subclade. H1avN2#E emerged in Brittany, the countrys most pig-dense region, and rapidly became the most frequently detected swIAV genotype across France. This drastic change in the swIAV lineages proportions at a national scale was unprecedented, making H1avN2#E a unique case for understanding swIAV evolution and spreading patterns. Phylogenetic analyses suggested an introduction of the H1avN2#E genotype from a restricted source, likely originating from Denmark. It spread rapidly with low genetic diversity at the start of the epizootic in 2020, showing increasing diversification in 2021 and 2022, and exhibiting reassortments with other enzootic genotypes. Amino acid sequence alignments of H1avN2#E antigenic sites revealed major mutations and deletions compared to vaccine 1C strain (HA-1C.2.2) and previously predominant H1avN1 strains (HA-1C.2.1). Antigenic cartography confirmed significant antigenic distances between H1avN2#E and other 1C strains, suggesting the new genotype escaped from the swine population preexisting immunity. Epidemiologically, the H1avN2#E virus exhibited epizootic hallmarks with more severe clinical outcomes compared to H1avN1 viruses. These factors likely contributed to the spread of H1avN2#E within the pig population. The rapid rise of H1avN2#E highlighted the dynamic nature of swIAV genetic and antigenic diversity, underscoring the importance of adapted surveillance programs to support risk assessment in the event of new outbreaks. This also demonstrate the need to strengthen biosecurity measures when receiving pigs in a herd and to limit trading of swIAV-excreting live swine between European countries.

evolutionary biology↗

bioRxiv2024

Induced degradation of SNAP-fusion proteins

Self-labeling protein tags are an efficient means to visualize, manipulate, and isolate engineered fusion proteins with suitable chemical probes. The SNAP-tag, which covalently conjugates to benzyl-guanine and -chloropyrimidine derivatives is used extensively in fluorescence microscopy, given the availability of suitable SNAP-ligand-based probes. Here, we extend the applicability of the SNAP-tag to targeted protein degradation. We developed a set of SNAP PROteolysis TArgeting Chimeras (SNAP-PROTACs), which recruit the VHL or CRBN-ubiquitin E3 ligases to induce the degradation of SNAP-fusion proteins. Endogenous tagging enabled the visualization and the selective depletion of a SNAP-clathrin light chain fusion protein using SNAP-PROTACs. The addition of PROTACs to the SNAP-tag reagent toolbox facilitates the comprehensive analysis of protein function with a single gene tagging event.

cell biology↗

bioRxiv2023

Micro- to nano-scale investigation of Precambrian metasediments: biogenicity and preservation in the 3.22 Ga Moodies Group (Barberton Greenstone Belt, S. Africa) and the 2.46 Ga Brockman Iron Formation (Hamersley Basin, W. Australia).

Precambrian metasediments provide a unique archive for understanding Earths earliest biosphere, however traces of microbial life preserved in ancient rocks are often controversial. In this study we leveraged several micro- to nano-scale techniques to study filamentous structures previously reported in clastic sediments of the 3.22 Ga Moodies Group, Barberton Greenstone Belt, S. Africa. We performed petrographic, mineralogical, electron microprobe, confocal fluorescence and electron microscopy analyses of these structures in order to evaluate their biogenicity and syngenecity. We also examined drill core samples of deep-water iron formations from the 2.46 Ga Joffre member of the Brockman Iron Formation (Hamersley Basin, W. Australia) to better understand their potential biogenicity. In both cases, we aimed to resolve primary vs. secondary mineral assemblages and their relation to filamentous or sedimentary structures. In the Moodies Group samples, filamentous structures were resolved by confocal imaging and revealed to be crosscut by later metamorphic phases, highlighting their syngenetic nature. Three-dimensional imaging reveals that while the filamentous structures are not necessarily associated with grain boundaries (e.g., as organic coatings), they form both sheets and filaments, complicating their interpretation but not ruling out a biological origin. No organic microstructures appeared to be preserved in our Dales Gorge samples. We also examined the possible application of electron paramagnetic resonance spectroscopy (EPR) to carbonaceous matter in ancient silica-rich matrices, similar to Bourbin et al. (2013), using samples from the Brockman iron formation. While resonance associated with organic matter was largely unresolvable in the Brockman iron formation samples due to their low organic matter contents, large effects on the EPR spectra were apparent stemming from the presence of magnetic iron minerals, highlighting the need to carefully consider sample composition in EPR analyses targeting ancient organic matter. Collectively, this study highlights the added value of micro- to nano-scale techniques as applied to Precambrian metasediments containing traces of ancient life, for example in revealing the pre-metamorphic emplacement and three-dimensional structure of filaments in the Moodies Group, but also the potential drawbacks and pitfalls, such as the case of strong magnetic mineral interference in EPR analysis of organic matter in trace abundance in the Dales Gorge.

paleontology↗

bioRxiv2022

Proteomic discovery of chemical probes that perturb protein complexes in human cells

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

biochemistry↗

bioRxiv2022

Assigning functionality to cysteines by base editing of cancer dependency genes

Chemical probes are lacking for most human proteins. Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse proteins. Determining which of these covalent binding events impact protein function, however, remains challenging. Here, we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cell proliferation. We show that the resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, correctly predicts the essentiality of cysteines targeted by cancer therapeutics and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines on >110 cancer dependency proteins. We further demonstrate how measurements of reactivity in native versus denatured proteomes can discriminate essential cysteines amenable to chemical modification from those buried in protein structures, providing a valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

systems biology↗

bioRxiv2022

The metabolite alpha-ketobutyrate increases health and life spans by activating AMPK

Aging is a complex process that is directly related to human health and disease. The extraordinary finding that aging is malleable, as shown in model organisms whose life and health spans are extended by specific gene mutations or dietary or pharmacological perturbations 1-3, has offered enormous hope for our understanding and treatment of aging and related diseases. Although many molecules have been identified that can extend the lifespan of model organisms, few have been shown to alleviate age-related symptoms or illness in mammals 4. Here we show that supplementation with the endogenous metabolite -ketobutyrate (-KB) increases the lifespan of adult C. elegans. Using Gelfree DARTS-PROTOMAP, we identified microtubule-actin cross-linking factor (MACF1) that was protected against proteolysis in the presence of -KB. MACF1 belongs to the spectraplakin family of giant, evolutionarily conserved proteins with versatile functions 5, but their link to longevity regulation has not been explored. -KBs longevity effect in C. elegans is abrogated by loss-of-function mutation in vab-10, encoding the worm ortholog of mammalian MACF1 6. Like -KB treatment, vab-10 knockdown activates AMP-activated protein kinase (AMPK), and AMPK is required for -KB effects on longevity. The findings suggest a model in which -KB increases longevity by activating AMPK via VAB-10/MACF1 modulation. -KB also delays aging in mammals, increasing the lifespan of aged male mice and the healthspan of both male and female animals. Targeting of broadly expressed scaffolding proteins in connection to cellular energy homeostasis seems to be a clever way that nature has devised for metabolite signals to impinge upon multiple organ and tissue systems, which may have utility for controlling aging and related diseases.

physiology↗

bioRxiv2020

Dissecting signalling hierarchies in the patterning of the mouse primitive streak using micro-patterned EpiLC colonies.

Molecular embryology studies have established that the patterning of the gastrula-stage mouse embryo is dependent on a regulatory network where the WNT, BMP and NODAL signalling pathways cooperate. Still, important aspects of their respective contributions to this process remain unclear. Here, studying their impact on the spatial organization and the developmental trajectories of micro-patterned Epiblast Like Cells (EpiLC) colonies, we show that when BMP is present, it dominates NODAL and WNT and imposes a posterior character to the colonies differentiation. However, the use of two Nodal mutant cell lines allowed us to show that prior to BMP action, NODAL is required to establish the mesendodermal lineage. The fact that mutant phenotypes were more severe in vitro than in vivo suggests that embryonic phenotypes are partially rescued by ligands of extra-embryonic or maternal origin. Our work demonstrates the complementarity of micro-patterned EpiLC colonies to embryological approaches.

developmental biology↗

bioRxiv2020

Development of a soft cell confiner to decipher the impact of mechanical stimuli on cells

Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all tested cell lines. The soft cell confiner is thus a valuable tool to decipher the effect of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY A unique tool to analyze the role of long-term effect of mechanical confinement in normal and pathological conditions

biophysics↗

bioRxiv2019

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving direct functional perturbation and/or ligand-induced degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells, underscoring the potential of electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

immunology↗

bioRxiv2019

Simultaneous tracking of division and differentiation from individual hematopoietic stem and progenitor cells reveals within-family homogeneity despite population heterogeneity

The advent of high throughput single cell methods such as scRNA-seq has uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs). A significant issue is how to reconcile those findings with the standard model of hematopoietic development, and a fundamental question is how much instruction is inherited by offspring from their ancestors. To address this, we further developed a high-throughput method that enables simultaneously determination of common ancestor, generation, and differentiation status of a large collection of single cells. Data from it revealed that while there is substantial population-level heterogeneity, cells that derived from a common ancestor were highly concordant in their division progression and share similar differentiation outcomes, revealing significant familial effects on both division and differentiation. Although each family diversifies to some extent, the overall collection of cell types observed in a population is largely composed of homogeneous families from heterogeneous ancestors. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell-surface markers that are used for phenotypic HSPC identification: CD48, SCA-1, c-kit and Flt3. These data call for a revision of the fundamental model of haematopoiesis from a single tree to an ensemble of trees from distinct ancestors where common ancestor effect must be considered. As HSPCs are cultured in the clinic before bone marrow transplantation, our results suggest that the broad range of engraftment and proliferation capacities of HSPCs could be consequences of the heterogeneity in their engrafted families, and altered culture conditions might reduce heterogeneity between families, possibly improving transplantation outcomes.

immunology↗

bioRxiv2017

DNA-PK facilitates HIV transcription by regulating the activity of RNA polymerase II and the recruitment of transcription machinery at HIV LTR

Despite the use of highly effective antiretroviral therapy (HAART), the presence of latent or transcriptionally silent proviruses prevents cure and eradication of HIV infection. These transcriptionally silent proviruses are well protected from both the immune system and HAART regimens. Thus, in order to tackle the problem of latent HIV reservoirs, it is a prerequisite to define all the pathways that regulate HIV transcription. We have previously reported that DNA-PK facilitates HIV transcription by interacting with the RNA polymerase II (RNAP II) complex recruited at HIV LTR. To extend those studies further, here we demonstrate that DNA-PK promotes HIV transcription by supporting it at several stages, including initiation, pause-release and elongation. We discovered that DNA-PK increases phosphorylation of RNAP II C-terminal domain (CTD) at serine 5 (Ser5) and serine 2 (Ser2) by both directly catalyzing and by augmenting the recruitment of P-TEFb at HIV LTR. We found that DNA-PK facilitates the establishment of euchromatin structure at HIV LTR, which further supports HIV gene expression. DNA-PK inhibition or knockdown leads to the severe impairment of HIV gene expression and conversion of euchromatin to heterochromatin at HIV LTR. It also profoundly restricts HIV replication and reactivation of latent provirus. DNA-PK promotes the recruitment of TRIM28 at LTR and facilitates the release of paused RNAP II through TRIM28 phosphorylation. The results were reproduced in cell lines belonging to both lymphoid and myeloid lineages and were confirmed in primary CD4+ T cells and peripheral blood mononuclear cells (PBMCs) from HIV-infected patients.\n\nIMPORTANCEOur results reveal the important role of DNA-PK in supporting HIV transcription, replication and latent proviral reactivation. Intriguingly, this study sheds light on an important pathway that affects HIV gene expression. These findings provide strong rationale for developing and using transcriptional inhibitors, such as DNA-PK inhibitors, as supplement to HAART regimens in order to further enhance their effectiveness and to suppress toxicity due to HIV proteins.

molecular biology↗

bioRxiv2017

CBF-1 promotes the establishment and maintenance of HIV latency by recruiting Polycomb repressive complexes, PRC1 and PRC2, at HIV LTR

The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the HIV-1 LTR promoter. Here we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in the reactivation of latent HIV proviruses. Chromatin immunoprecipitation (ChIP) assays using latently infected primary T cells and Jurkat T-cell lines demonstrated that CBF-1 induces the establishment and maintenance of HIV latency by recruiting Polycomb Group (PcG/PRC) corepressor complexes or Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Knockdown of CBF-1 resulted in the dissociation of PRCs corepressor complexes enhancing the recruitment of RNA polymerase II (RNAP II) at HIV LTR. Knockdown of certain components of PRC1 and PRC2 also led to the reactivation of latent proviruses. Similarly, treatment of latently infected primary CD4+ T cells with the EZH2 inhibitor, 3-deazaneplanocin A (DZNep), led to their reactivation.

molecular biology↗