Biology subjects

Guillonneau, F.

Publications and source records attributed to Guillonneau, F..

10 recordsLinked to original sources

bioRxiv2026

Molecular glue degraders of CK1α remodel lysosomes and limit ferroptosis in glioblastoma stem-like cells

Glioblastoma stem-like cells (GSCs) are highly plastic tumor cells that drive initiation, progression and therapeutic resistance. Although lysosomal homeostasis has emerged as a critical determinant of GSC fitness, the molecular mechanisms regulating this compartment remain incompletely understood. Here, using complementary proteomic approaches, we identified a lysosomal signature associated with the kinase CK1 in THP1 monocytic cells. Biochemical analyses revealed that genetic or siRNA-mediated depletion of CK1 altered the overall abundance and maturation of late endosome-lysosome-associated proteins in both THP1 cells and two patient-derived GSCs. This lysosomal remodeling was associated with impaired tumorsphere formation capacity in GSCs. To further explore the therapeutic potential of targeting CK1, we employed selective molecular glue degraders (MGDs). Acute CK1 degradation efficiently recapitulated lysosomal remodeling and impaired tumorsphere formation in GSCs. While CK1-targeted MGDs did not sensitize GSCs to lysosomal membrane permeabilization induced by L-leucyl-L-leucine methyl ester (LLOMe), they protected from GPX4 inhibitor-induced ferroptosis. Together, these findings identify CK1 as a regulator of lysosomal fitness and ferroptotic sensitivity, suggesting a connection between CK1-dependent lysosome remodeling and associated cell death pathways.

cancer biology↗

bioRxiv2026

Desmin p.R406W mutation is associated with arrhythmias through structural and electrophysiological remodeling

Background and AimsMutations in the desmin (DES) gene cause a variety of cardiomyopathies associated with arrhythmias, yet the electrophysiological consequences of these variants remain largely uncharacterized. The aim of this study was to investigate the pathogenic mechanisms of the de novo DES p.R406W variant, which was identified in a 9-year-old patient who suffered from severe ventricular arrhythmias and sudden cardiac death without overt structural heart disease. MethodsHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the DES p.R406W variant (including the patients line) were compared to isogenic controls. Action potentials (AP) of hiPSC-CMs were recorded using patch-clamp. Furthermore, 3D engineered heart tissues (EHTs) were generated from hiPSC-CMs and their APs were recorded with sharp microelectrodes. Analytical techniques also included transmission electron microscopy (TEM) and integrated transcriptomic and proteomic profiling. Finally, a heterozygous knock-in (KI) mouse model carrying the Des p.R405W ortholog was evaluated through surface ECG, echocardiography and ex vivo cardiac optical mapping. ResultsThe DES p.R406W mutation prolonged AP duration in IM-R406W hiPSC-CMs and EHTs vs Control ones. Multi-omics analysis of EHTs revealed a dysregulation of genes and proteins involved in contractile function, cell adhesion, and electrical activity. TEM imaging revealed changes in Z-disc architecture in mutant tissues. Twenty-week-old Des p.R405W KI mice exhibited ventricular conduction slowing (prolonged QRS) and a high susceptibility to ventricular tachyarrhythmias, likely due to reentrant mechanisms. Mild hypertrophy was also observed, but only in females. ConclusionThe DES p.R406W variant is highly pathogenic, causing electrical and structural remodeling of the myocardium. This study highlights the effectiveness of hiPSC-CMs and EHTs in recapitulating the clinical phenotype of desminopathy, providing a platform for investigating the mechanisms of early-onset cardiac arrhythmias and SCD.

pathology↗

bioRxiv2025

EGFR/TBK1-dependent mitochondrial quality control contributes to acquired resistance to temozolomide

Acquisition of secondary resistance to chemotherapy in cancer cells is an ill-characterized multi-step process. We conducted a non-supervised, global proteomic analysis of U251 glioma cells treated by temozolomide as they transit from drug-sensitive to tolerant and then resistant states. This unprecedented analysis revealed key mitochondrial evolutions throughout the process. Following an early stress substantiated by a transient peak of reactive oxygen species, mitochondrial quality control mechanisms enable emergence of the resistant population. We combined genome editing, targeted protein degradation via PROTAC, and pharmacologic inhibitors to investigate the mechanisms underlying stress resolution both at population and single cell scales (including microscopic evaluation and in-situ proximity ligation assays). These studies unravel a Src-activated EGFR and TBK1 axis that initiates autophagic recycling of mitochondria as a necessary step to the emergence of the resistant population.

cancer biology↗

bioRxiv2025

The Kinase CK1α coordinates Initiation and Termination of the cGAS-STING Pathway

The cGAS-STING pathway is an evolutionarily conserved antimicrobial defense mechanism that senses cytosolic DNA to trigger innate immune responses. cGAS and STING play dual roles in tumorigenesis, promoting antitumor immunity and cell death while fueling tumor growth and metastasis. However, the mechanisms fine-tuning this pathway remain elusive. Using proteomic approaches, we report that Casein Kinase 1 alpha (CK1) operates as a bimodal regulator of the cGAS-STING pathway. CK1 supports optimal DNA sensing by preventing the proteasomal degradation of cGAS driven by the cullin-RING ubiquitin ligase 3 (CRL3). Conversely, CK1 facilitates STING degradation and signaling termination in response to STING agonists, tempering IRF3 activation. Exploiting these counterposing functions, we show that selective degradation of CK1 with molecular-glue degraders impaired the survival of a triple-negative breast cancer cell line with chronic cGAS-STING activation and synergized with a STING agonist to kill acute myeloid leukemia cells. Thus, CK1s dual regulatory role in the cGAS-STING pathway presents a promising target for therapeutic development. TEASERThis study unveils CK1 as a bimodal regulator of the cGAS-STING pathway.

immunology↗

bioRxiv2025

MCL-1 as a molecular switch between myofibroblastic and pro-angiogenic features of breast cancer-associated fibroblasts

Breast cancer-associated fibroblasts (bCAFs) comprise inflammatory CAFs (iCAFs), characterized by the secretion of pro-inflammatory cytokines, and myofibroblastic CAFs (myCAFs), distinguished by their high production of extracellular matrix and their immunosuppressive properties. We previously showed that targeting the anti-apoptotic protein MCL-1 in primary culture of bCAF derived directly from human samples reduces their myofibroblastic characteristics. We herein show by single-cell RNA-sequencing analysis of bCAFs that MCL-1 knock down induces a phenotypic shift from wound-myCAF to IL-iCAF, characterized by the upregulation of genes associated with inflammation as well as angiogenesis-related genes. In vitro, genetic and pharmacologic MCL-1 inhibition increases VEGF secretion by bCAFs, enhancing endothelial cell tubulogenesis. In a chicken chorioallantoic membrane (CAM) model in ovo, co-engraftment of breast cancer cells and bCAFs with reduced MCL-1 expression leads to heightened peritumoral vascular density, driven by VEGF. Mechanistically, the pro-angiogenic phenotype revealed by MCL-1 inhibition is dependent on BAX-BAK activity. It results in NF-{kappa}B activation, inhibition of which by a IKK{beta} inhibitor suppresses the transcription of VEGF and pro-inflammatory factors triggered by MCL-1 inhibition in bCAFs. Chemotherapy induces a downregulation of MCL-1 in bCAFs and it promotes a pro-angiogenic phenotype, counteracted by overexpressed MCL-1. Overall, these findings uncover a novel regulatory function of MCL-1 in determining bCAF subpopulation differentiation and highlight its role in modulating their pro-angiogenic properties, in response to treatment in particular.

cancer biology↗

bioRxiv2023

NOXA/MCL-1 axis determines cell-death decision between apoptosis and pyroptosis and the inflammatory secretome of breast cancer cells treated with anti-mitotics.

Understanding how the malignant cells respond to chemotherapy is essential to prevent the development of resistance and to improve the efficiency of anti-cancer drugs. Recently, we established that, by intrinsic and paracrine mechanisms, taxol treatment in breast tumor cells increases NOXA a pro-apoptotic protein functioning as an endogenous inhibitor of survival protein MCL-1, thereby enhancing cytotoxic load on the compensatory survival protein BCL-xL. We herein sought to define the contribution of NOXA/MCL-1 to the modality of cell death secretome composition upon anti-mitotic treatment associated with a BCL-xL antagonist. We observed that genetic inactivation of NOXA (enforcing MCL-1 pro-survival activity) in cancer cells not only delays their death when exposed to taxol in combination with the BCL-xL antagonist A1331852, but also alters its morphological characteristics with the apparition of features evoking pyroptosis. We identified the Caspase3-GSDME axis as regulating pyroptotic-like features suggesting that NOXA may act as a negative regulator of this cell death process (and MCL-1 as a positive regulator for it). Furthermore, comparative analysis of secretomes from the NOXA proficient or deficient cancer cells treated by taxol reveals variations in inflammatory cytokine production including those of IL-1{beta} and IL-18. Thus, our results show that anti-mitotic treatments are able to induce death by apoptosis and/or pyroptosis depending on BCL-2 family balance in breast cancer cells. Furthermore, NOXA/MCL-1 ratio appears to control the communication between these two types of cell death and their associated extracellular inflammatory signals in coordination with the pore-forming gasdermin GSDME.

cancer biology↗

bioRxiv2020

USP9X deubiquitinase couples the pluripotency network and cell metabolism to regulate ESC differentiation potential

Embryonic stem cells (ESC) have the unique ability to differentiate into all three germ cell layers. ESC transition through different states of pluripotency in response to growth factor signals and environmental cues before becoming terminally differentiated. Here, we demonstrated, by a multi-omic strategy, that the deubiquitinase USP9X regulates the developmental potential of ESC, and their transition from a naive to a more developmentally advance, or primed, state of pluripotency. We show that USP9X facilitates developmental gene expression and induces modifications of the mitochondrial bioenergetics, including decreased routing of pyruvate towards its oxidation and reduced respiration. In addition, USP9X binds to the pluripotency factor ESRRB, regulates its abundance and the transcriptional levels of a subset of its target genes. Finally, under permissive culture conditions, depletion of Usp9X accelerates cell differentiation in all cell lineages. We thus identified a new regulator of naive pluripotency and show that USP9X couples ESRRB pluripotency transcriptional network and cellular metabolism, both of which are important for ESC fate and pluripotency.

cell biology↗

bioRxiv2019

Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts

DNA hemicatenanes (HCs) are DNA structures in which one strand of a double stranded helix passes through the two strands of another double stranded DNA. Frequently mentioned as DNA replication, recombination and repair intermediates, they have been proposed to participate in the spatial organization of chromosomes and in the regulation of gene expression. To explore potential roles of HCs in genome metabolism, proteins capable of binding specifically HCs were purified by fractionating nuclear extracts from Hela cells. This approach identified three RNA-binding proteins: the Tudor-Staphylococcal Nuclease Domain 1 (SND1) protein and two proteins from the Drosophila behavior human splicing family, the ParaSpeckle Protein Component 1 and the Splicing Factor Proline- and Glutamine- rich protein. Since these proteins were partially pure after fractionation, truncated forms of these proteins were expressed in E. coli and purified to near homogeneity. The specificity of their interaction with HCs was re-examined in vitro. The two truncated purified SND1 proteins exhibited a high specificity for HCs, suggesting a role of SND1 protein in targeting the basic transcription machinery to HC structures.

biochemistry↗

bioRxiv2018

Genomic from Plasmodium falciparum field isolates from Benin allows the identification of PfEMP1 variants by LC-MS/MS at the patient’s level

PfEMP1 are the major protein family from parasitic origin involves in the pathophysiology of severe malaria, and PfEMP1 domain subtypes are associated with the infection outcome. In addition, PfEMP1 variability in endless and current protein repository do not reflect the immense diversity of the sequences of PfEMP1 proteins. The aim of our study was to identify the different PfEMP1 variants expressed within a patient sample by mass spectrometry. We performed a proteogenomic approach to decipher at the patients level PfEMP1 expression in different clinical settings: cerebral malaria, severe anemia and uncomplicated malaria. The combination of whole genome sequencing approach, RNAsequencing, and mass spectrometry proteomic analysis allowed to attribute PfEMP1 sequences to each sample and classify the relative expression level of PfEMP1 proteins within each sample. We predicted PfEMP1 structures using the newly identified protein sequences. We confirmed the involvement of DBL{beta} in malaria pathogenesis and observed that CIDR domains linked to ICAM-1 binding DBL{beta} domains displayed EPCR binding structure.

microbiology↗

bioRxiv2018

Deep proteomic analysis of chicken erythropoiesis

In contrast to mammalian erythroid cells that lost their nucleus at the end of the differentiation process, circulating chicken erythrocytes, like erythrocytes of most other non-mammalian vertebrates, are nucleated although their nucleus is believed to be transcriptionally silent. This major difference suggests that the erythroid differentiation process is likely to present both similarities and differences in mammals compared to other vertebrates. Since proteins are the major cellular effectors, analysis of the proteome is more prone to reflect true differences than analysis of the pattern of mRNA expression. We have previously reported the evolution of the proteome of human erythroid cells throughout their differentiation process. Here we report the analysis of the proteome of chicken erythroblasts during their terminal differentiation. We used the T2EC cellular model that allows to obtain homogenous populations of immature erythroblasts. Induction of their terminal differentiation led to their maturation and the possibility to obtain cells at different differentiation stages. Mass spectrometry analysis of these cell populations allowed the absolute quantification of 6167 proteins throughout the terminal differentiation process. Beside many proteins with similar expression patterns between chicken and human erythroblasts, like SLC4A1 (Band3), GATA1 or CD44, this analysis also revealed that other important proteins like Kit or other GATA transcription factors exhibit fully different patterns of expression.

developmental biology↗