Publications

2024

• NHSL3 controls single and collective cell migration through two distinct mechanisms
  
  • Novikov Nikita M
  • Gao Jinmei
  • Fokin Artem I
  • Rocques Nathalie
  • Chiapetta Giovanni
  • Rysenkova Karina
  • Zea Diego Javier
  • Polesskaya Anna
  • Vinh Joelle
  • Guérois Raphaël
  • Gautreau Alexis
  Nature Communications, Nature Publishing Group, 2024. The molecular mechanisms underlying cell migration remain incompletely understood.<p>Here, we show that knock-out cells for NHSL3, the most recently identified member of the Nance-Horan Syndrome family, are more persistent than parental cells in single cell migration, but that, in wound healing, follower cells are impaired in their ability to follow leader cells. The NHSL3 locus encodes several isoforms. We identify the partner repertoire of each isoform using proteomics and predict direct partners and their binding sites using an AlphaFold2-based pipeline. Rescue with specific isoforms, and lack of rescue when relevant binding sites are mutated, establish that the interaction of a long isoform with MENA/VASP proteins is critical at cell-cell junctions for collective migration, while the interaction of a short one with 14-3-3θ in lamellipodia is critical for single cell migration. Taken together, these results demonstrate that NHSL3 regulates single and collective cell migration through distinct mechanisms.</p> (10.1038/s41467-024-55647-3)
  DOI : 10.1038/s41467-024-55647-3
• Characterization of a novel variant in the NR3C1 gene: differentiating glucocorticoid resistance from Cushing Syndrome
  
  • Laulhé Margaux
  • Yacobi Bach Michal
  • Perrot Julie
  • Gershinsky Michal
  • Fagart Jérôme
  • Shefer Gabi
  • Amazit Larbi
  • Kamenický Peter
  • Viengchareun Say
  • Martinerie Laetitia
  • Greenman Yona
  Journal of Clinical Endocrinology and Metabolism, Endocrine Society, 2024. Abstract Introduction Primary generalized glucocorticoid resistance syndrome (GGRS) is a rare endocrine disease caused by loss-of-function variants of the NR3C1 gene encoding the Glucocorticoid Receptor. We describe a novel heterozygous missense variant (NM_000176.3, c.1330T&gt;G, p.Phe444Val) within the DNA Binding Domain. Clinical case Elevated urinary-free cortisol levels were detected in a 59-year-old male before bariatric surgery (BMI 39.9 kg/m2). Early-onset hypertension was well controlled. The low dose dexamethasone suppression test was pathologic, but ACTH and midnight salivary cortisol levels were normal. The patient was initially referred to transsphenoidal surgery for a presumed diagnosis of Cushing disease. He presented to our department at the age of 68, when the clinical diagnosis of GGRS was established. Methods Functional characterization of the variant was performed ex vivo through transient transfection assays in HEK 293T cells to assess transcriptional activity and nuclear translocation. Results The variant showed a lack of transcriptional activity (GRWT: 91.5 [80.5; 101.2] vs. GRF444V: 1.0 [1.0; 1.0]) despite efficient nuclear translocation in response to dexamethasone, suggesting a DNA binding defect of the variant. These results are discussed in the light of previously reported GGRS cases. Conclusion We have described a novel heterozygous mutation of the NR3C1 gene associated with primary GGRS. This case highlights the importance of raising awareness of clinical and laboratory features of this rare disorder, to enable early diagnosis and avoid unnecessary and potentially dangerous diagnostic and therapeutic procedures. (10.1210/clinem/dgae829)
  DOI : 10.1210/clinem/dgae829
• Vinculin–Arp2/3 interaction inhibits branched actin assembly to control migration and proliferation
  
  • James John
  • Fokin Artem I
  • Guschin Dmitry Y
  • Wang Hong
  • Polesskaya Anna
  • Rubtsova Svetlana N
  • Clainche Christophe Le
  • Silberzan Pascal
  • Gautreau Alexis M
  • Romero Stéphane
  Life Science Alliance, Life Science Alliance LLC, 2024, 8 (2), pp.e202402583. Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosinmediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here, we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI-P878A) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density. (10.26508/lsa.202402583)
  DOI : 10.26508/lsa.202402583
• RNase W, a conserved ribonuclease family with a novel active site
  
  • Vayssières Marlène
  • Jüttner Michael
  • Haas Karina
  • Ancelin Aurélie
  • Marchfelder Anita
  • Leulliot Nicolas
  • Ferreira-Cerca Sébastien
  • Blaud Magali
  Nucleic Acids Research, Oxford University Press, 2024, 29 (8), pp.1255-1273. Abstract Ribosome biogenesis is a complex process requiring multiple precursor ribosomal RNA (rRNA) cleavage steps. In archaea, the full set of ribonucleases (RNases) involved in rRNA processing remains to be discovered. A previous study suggested that FAU-1, a conserved protein containing an RNase G/E-like protein domain fused to a domain of unknown function (DUF402), acts as an RNase in archaea. However, the molecular basis of this activity remained so far elusive. Here, we report two X-ray crystallographic structures of RNase G/E-like–DUF402 hybrid proteins from Pyrococcus furiosus and Sulfolobus acidocaldarius, at 2.1 and 2.0 Å, respectively. The structures highlight a structural homology with the 5′ RNA recognition domain of Escherichia coli RNase E but no homology with other known catalytic nuclease domains. Surprisingly, we demonstrate that the C-terminal domain of this hybrid protein, annotated as a putative diphosphatase domain, harbors the RNase activity. Our functional analysis also supports a model by which the RNase G/E-like domain acts as a regulatory subunit of the RNase activity. Finally, in vivo experiments in Haloferax volcanii suggest that this RNase participates in the maturation of pre-16S rRNA. Together, our study defines a new RNase family, which we termed the RNase W family, as the first archaea-specific contributor to archaeal ribosome biogenesis. (10.1093/nar/gkae907)
  DOI : 10.1093/nar/gkae907
• Structure of the Nmd4-Upf1 complex supports conservation of the nonsense-mediated mRNA decay pathway between yeast and humans
  
  • Barbarin-Bocahu Irène
  • Ulryck Nathalie
  • Rigobert Amandine
  • Ruiz Gutierrez Nadia
  • Decourty Laurence
  • Raji Mouna
  • Garkhal Bhumika
  • Le Hir Hervé
  • Saveanu Cosmin
  • Graille Marc
  PLoS Biology, Public Library of Science, 2024, 22 (9), pp.e3002821. The nonsense-mediated mRNA decay (NMD) pathway clears eukaryotic cells of mRNAs containing premature termination codons (PTCs) or normal stop codons located in specific contexts. It therefore plays an important role in gene expression regulation. The precise molecular mechanism of the NMD pathway has long been considered to differ substantially from yeast to metazoa, despite the involvement of universally conserved factors such as the central ATP-dependent RNA-helicase Upf1. Here, we describe the crystal structure of the yeast Upf1 bound to its recently identified but yet uncharacterized partner Nmd4, show that Nmd4 stimulates Upf1 ATPase activity and that this interaction contributes to the elimination of NMD substrates. We also demonstrate that a region of Nmd4 critical for the interaction with Upf1 in yeast is conserved in the metazoan SMG6 protein, another major NMD factor. We show that this conserved region is involved in the interaction of SMG6 with UPF1 and that mutations in this region affect the levels of endogenous human NMD substrates. Our results support the universal conservation of the NMD mechanism in eukaryotes. (10.1371/journal.pbio.3002821)
  DOI : 10.1371/journal.pbio.3002821
• RNA methyltransferase holoenzymes : implications in mRNA maturation and translation and in neurodevelopmental disorders
  
  • Graille Marc
  , 2024. Modified nucleotides in non-coding RNAs, such as rRNAs, tRNAs and snRNAs, represent an important layer of gene expression regulation through their ability to fine-tune mRNA maturation and translation. Dysregulation of such modifications and the enzymes installing them have been linked to various human pathologies including neurodevelopmental disorders and cancers. Several methyltransferases (MTases) are regulated allosterically by human TRMT112. The complete interactome of this regulator and the substrates of its interacting MTases have been only recently characterized, confirming that all these TRMT112-MTase complexes modify factors (snRNAs, tRNAs, rRNAs and proteins) involved in the mRNA maturation and translation processes. Indeed, the TRMT112-BUD23 and TRMT112-METTL5 complexes catalyze the formation of N7-methylguanosine (m7G) and N6-methyladenosine (m6A) on the 18S rRNA and participate in 40S ribosomal subunit biogenesis pathway. The TRMT112-HEMK2 complex modifies the translation termination factor eRF1, which is a tRNA mimicry critical for the release of the newly synthesized proteins. The TRMT112-ALKBH8, TRMT112-TRMT11 and TRMT112-THUMPD3 complexes contribute to translation elongation by modifying tRNAs. Finally, the TRMT112-THUMPD2 complex methylates U6 snRNA, the core component of the major catalytic spliceosome and hence is important for optimal splicing of weak splice sites. Recent results obtained on the functional characterization of eight homozygous METTL5 missense mutants identified in patients suffering from intellectual disability and microcephaly with varying degrees of penetrance will be presented. This work brings decisive information on the importance of this 18S rRNA m6A methylation, in normal brain development. Considering that the modification is adjacent to the ribosomal decoding site, it suggests that its absence rewires translation with important consequences in maturing neurons.
• A study of selected endocrine disrupting chemicals and their binding to host molecules with molecular modelling.
  
  • Mazurek Anna Helena
  , 2024. Endocrine Chemical Disruptors (EDCs) are substances that exhibit adverse effects as a consequence of an endocrine mode of action. It often includes interaction with receptors in the same way as receptor’s natural ligands. Among EDCs there are Active Pharmaceutical Ingredients (APIs) such as steroid hormones. Cyclodextrins (CDs) are cyclic oligosaccharides used as drug delivery systems for APIs of a low solubility in water, and as toxin removing agents. The goal of this study was to develop different molecular modelling techniques to analyze interactions between chosen EDCs and Estrogen Receptor (ER) or CDs. This could lead to a better understanding of the EDCs interactions with the chosen host molecules and possibly show a way to better recognize new potential EDCs. Moreover, the research could point out new direction for toxic EDCs removal or steroidal APIs delivery with application of CDs. The chosen EDCs were: estradiol (EST), progesterone (PRO), bisphenol A (BPA), two of them being APIs (EST and PRO), two of them being classic good examples of well-described EDCs (EST and BPA), one of them being a natural ligand of ER (EST) and one of them being an external to a human body toxin (BPA).Following molecular modelling methods have been in this study: 1) for the first time parametrization of chosen EDCs and CD in AMOEBA polarizable force field and succeeding Molecular Dynamics (MD) simulation of the ER + EST system using Tinker software, 2) benchmark tests of applied parameters in Quantum Mechanics (QM) (Density Functional Theory and semi-empirical approaches) and Molecular Mechanics (MD/MMGBSA) based computation approaches on the example of EST+β-CD system, 3) for the first time crystallization and structural analysis of the EST+β-CD system both in a crystalline state and as a water solution (both computational and experimental approaches).The results and conclusions are as follows: 1) MD simulations of crystal structures showed improved agreement with experiment over an additive force field. A 10 ns simulation of the EST-ER complex was stable and protein-hormone interactions wellreproduced. The developed parameters should thus be suitable for studying hormone and EDC interactions with their hosts. 2) This work for the first time justifies the EST-βCD molar ratio as 1:2 using the high-resolution mass spectrometry and phase solubility studies. While some of the QM methods properly indicated the correct host: guest ratio at the same time they failed to accurately predict the Gibbs free energy (ΔG) of complexation. QM methods that properly described the value of ΔG of 1:2 complex formation, failed to favouer it when compared with 1:1 complex ratio. MD/MMGBSA method accurately predicted the stability constant of the complexed but was not conclusive to indicate the formation of either 1:1 or 1:2 complex. 3) Using SCXRD, the crystal structure of the EST-βCD complex has been determined, being to the best of our knowledge the first solved crystal structure of an estrogen/CD complex. The periodic DFT calculations of NMR properties using GIPAW were found to be particularly helpful in the analysis of disorder in the solid state and interpretation of experimental NMR results
• Roles of NHSL3 in cell migration
  
  • Novikov Nikita
  , 2024. The molecular mechanisms underlying cell migration remain incompletely understood, despite many recent advances. Here, we show that NHSL3, a recently identified member of the Nance-Horan Syndrome protein family, is a novel regulator of single cell and collective migration. The NHSL3 gene, also known as KIAA1522, encodes as many as four isoforms through different transcription initiation sites and alternative splicing in human breast MCF10A cells. Using GFP fusion proteins, we observed that all three long isoforms were localized at the lamellipodium edge and at cell-cell contacts, whereas the short isoform was only present at the lamellipodium edge. The NHSL3 knock-out (KO), inactivating all isoforms in MCF10A cells, impaired migration of single cells and collective monolayers. When wounded, KO monolayers display decreased speed of the cells that follow leader cells when compared with parental cells, whereas single KO cells were more persistent than parental cells. We then rescued KO cells with the different isoforms. All isoforms more or less rescued the collective migration phenotype, the most efficient being isoform i2 and the least efficient being the short isoform i3S. In sharp contrast and to our surprise, only the short isoform i3S rescued single cell migration. Moreover, a specific siRNA that targets specifically the short isoform displayed the same migration phenotype in single cells as the NHSL3 KO that inactivates all isoforms.To get an insight into the mechanisms involved, we performed comparative proteomics of the different isoforms and sorted potential direct partners by a bioinformatics pipeline based on AlphaFold2 that predicted putative binding sites. Long isoforms of NHSL3 interacted with well-known regulators of actin cytoskeleton, such as WAVE regulatory complex (WRC), IRSp53 and MENA/VASP proteins. The short i3S isoform interacted with much fewer proteins, none of which being an obvious candidate to control migration persistence of single cells. In collective migration assays, a mutant form of NHSL3 i2 that does not bind to MENA/VASP proteins impaired the recruitment of MENA/VASP proteins to cell-cell contacts and did not rescue the collective migration phenotype of NHSL3 KO cells. In single cell assays, a mutant form of NHSL3 i3S designed to impair the interaction with the 14-3-3θ partner failed to recruit 14-3-3θ at the lamellipodium edge and did not rescue the single cell migration. Altogether these results demonstrate that NHSL3 regulates both single cell and collective migration through different mechanisms that involve specific interactions with 14-3-3θ and MENA/VASP proteins, respectively.
• The Arp2/3 inhibitory protein Arpin inhibits homology‐directed DNA repair
  
  • Simanov Gleb
  • Rocques Nathalie
  • Romero Stéphane
  • de Koning Leanne
  • Vacher Sophie
  • Dubois Thierry
  • Bièche Ivan
  • Gautreau Alexis
  Biology of the Cell, Wiley, 2024. Abstract Background information Arpin, an Arp2/3 inhibitory protein, inhibits lamellipodial protrusions and cell migration. Arpin expression is lost in tumor cells of several cancer types. Results Here we analyzed expression levels of Arpin and various markers using Reverse Phase Protein Array (RPPA) in human mammary carcinomas. We found that Arpin protein levels were correlated with those of several DNA damage response markers. Arpin‐null cells display enhanced clustering of double stand breaks (DSBs) when cells are treated with a DNA damaging agent, in line with a previously described role of the Arp2/3 complex in promoting DSB clustering for homologous DNA repair (HDR) in the nucleus. Using a specific HDR assay, we further showed that Arpin depletion increased HDR efficiency two‐fold through its ability to inactivate the Arp2/3 complex. Conclusions Arpin regulates both cell migration in the cytosol and HDR in the nucleus. Significance Loss of Arpin expression coordinates enhanced cell migration with up‐regulated DNA repair, which is required when DNA damage is induced by active cell migration. (10.1111/boc.202400073)
  DOI : 10.1111/boc.202400073
• Original Adverse Outcome Pathway linking neuronal exposure to nanoparticles to the onset of Alzheimer’s disease
  
  • Schneider Benoit
  , 2025, 20 (S1). Abstract Alzheimer’s disease (AD) is the most common dementia in humans that today concerns 50 million individuals worldwide and will affect more than 100 million people in 2050. Except for familial AD cases (&lt;5% of AD patients) for which AD pathology connects to mutations in critical genes involved in the processing of the amyloid precursor protein into neurotoxic Aß peptides, it remains unknown what provokes the overproduction and deposition of Aß peptides in the brain of sporadic AD cases (&gt;95% of AD patients). Some nanosized materials, e.g., nanoparticles (NPs), are suspected of playing a role in the growing incidence of AD, due to their reactivity with biological systems, easiness of crossing physiological barriers, capacity to reach the central nervous system and accumulate in the brain. Incriminated NPs are the ultrafine air‐borne particulate matter and manufactured NPs, such as titanium dioxide (TiO 2 ) NPs widely used in food and cosmetic industries or carbon black (CB) NPs used in rubber and as black pigment. Both TiO 2 ‐ and CB‐NPs display neurotoxicity, but the mechanisms by which those NPs affect neuronal homeostasis and place neurons on the path to degeneration are unknown. Combining in vitro and in vivo approaches, we provide prime evidence that TiO 2 ‐ and CB‐NPs bind a plasma membrane neuronal receptor, the cellular prion protein PrP C , well‐known for its implication in prion diseases, and corrupt PrP C signaling activity. Dysregulation of PrP signaling by TiO 2 ‐ or CB‐NPs pomotes the NADPH oxidase‐mediated overproduction of reactive oxygen species that alter neuronal redox equilibrium and the activation of 3‐phosphoinositide‐dependent kinase 1, at the root of an increased vulnerability of NP‐exposed neurons to inflammation and the overproduction of neurotoxic Aß peptides. By showing that neuronal exposure to some TiO 2 ‐ and CB‐NPs induce molecular signs of AD, these mechanistic data provide new insight into how human exposure to some engineered and environmental NPs may predispose to neurodegenerative diseases. Identifying the mechanisms by which nanoparticles and other pollutants alter neuronal homeostasis would enable the generation of a database of pollutant‐associated Adverse Outcome Pathways and provide clues for counteracting the adverse effects of those pollutants on the central nervous system. (10.1002/alz.087445)
  DOI : 10.1002/alz.087445
• Biogenèse des ribosomes et des ARN de transfert
  
  • Basta Tamara
  • Ferreira-Cerca Sébastien
  , 2024, pp.119-175. Ce chapitre décrit les connaissances sur la synthèse des ribosomes et des ARN de transfert chez les Archées. Les différentes étapes et les facteurs protéiques impliqués dans ces processus complexes sont détaillés. Enfin, le chapitre aborde les dernières avancées technologiques qui permettront de progresser dans la compréhension de ces processus fondamentaux chez les archées dans les années à venir. (10.51926/ISTE.9169.ch5)
  DOI : 10.51926/ISTE.9169.ch5
• Exposure to Selenomethionine and Selenocystine Induces Redox-Mediated ER Stress in Normal Breast Epithelial MCF-10A Cells
  
  • Dauplais Marc
  • Romero Stephane
  • Lazard Myriam
  Biological Trace Element Research, Humana Press, 2024. Selenium is an essential trace element co-translationnaly incorporated into selenoproteins with important biological functions. Health benefits have long been associated with selenium supplementation. However, cytotoxicity is observed upon excessive selenium intake. The aim of this study is to investigate the metabolic pathways underlying the response to the seleniumcontaining amino acids selenomethionine and selenocysteine in a normal human breast epithelial cell model. We show that both selenomethionine and selenocystine inhibit the proliferation of non-cancerous MCF-10A cells in the same concentration range as cancerous MCF-7 and Hela cells, which results in apoptotic cell death. Selenocystine exposure in MCF- 10A cells caused a severe depletion of free low molecular weight thiols, which might explain the observed up-regulation of the expression of the oxidative stress pathway transcription factor NRF2. Both selenomethionine and selenocystine induced the expression of target genes of the unfolded protein response (GRP78, ATF4, CHOP). Using a redox-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we show that both selenoamino acids shifted the ER redox balance towards an even more oxidizing environment. These results suggest that alteration of the redox state of the ER may disrupt protein folding and cause ER stress-induced apoptosis in MCF-10A cells exposed to selenoamino acids. (10.1007/s12011-024-04244-y)
  DOI : 10.1007/s12011-024-04244-y
• PI 3-Kinase and the Histone Methyl-Transferase KMT2D Collaborate to Induce Arp2/3-Dependent Migration of Mammary Epithelial Cells
  
  • Rysenkova Karina
  • Gaboriaud Julia
  • Fokin Artem
  • Toubiana Raphaëlle
  • Bense Alexandre
  • Mirdass Camil
  • Jin Mélissa
  • Ho Minh Chau N.
  • Glading Elizabeth
  • Vacher Sophie
  • Courtois Laura
  • Bièche Ivan
  • Gautreau Alexis
  Cells, MDPI, 2024, 13 (10), pp.876. Breast cancer develops upon sequential acquisition of driver mutations in mammary epithelial cells; however, how these mutations collaborate to transform normal cells remains unclear in most cases. We aimed to reconstitute this process in a particular case. To this end, we combined the activated form of the PI 3-kinase harboring the H1047R mutation with the inactivation of the histone lysine methyl-transferase KMT2D in the non-tumorigenic human mammary epithelial cell line MCF10A. We found that PI 3-kinase activation promoted cell-cycle progression, especially when growth signals were limiting, as well as cell migration, both in a collective monolayer and as single cells. Furthermore, we showed that KMT2D inactivation had relatively little influence on these processes, except for single-cell migration, which KMT2D inactivation promoted in synergy with PI 3-kinase activation. The combination of these two genetic alterations induced expression of the ARPC5L gene that encodes a subunit of the Arp2/3 complex. ARPC5L depletion fully abolished the enhanced migration persistence exhibited by double-mutant cells. Our reconstitution approach in MCF10A has thus revealed both the cell function and the single-cell migration, and the underlying Arp2/3-dependent mechanism, which are synergistically regulated when KMT2D inactivation is combined with the activation of the PI 3-kinase. (10.3390/cells13100876)
  DOI : 10.3390/cells13100876
• InsPection of electron density maps supports wrongly modeled hexakisphosphate (InsP6) bound to African swine fever mRNA-decapping enzyme g5Rp
  
  • Graille Marc
  Journal of Virology, American Society for Microbiology, 2024, 98 (5). (10.1128/jvi.01597-23)
  DOI : 10.1128/jvi.01597-23
• A novel mutation in the NR3C1 gene associated with reversible glucocorticoid resistance
  
  • Laulhé Margaux
  • Kuhn Emmanuelle
  • Bouligand Jérôme
  • Amazit Larbi
  • Perrot Julie
  • Lebigot Elise
  • Kamenickỷ Peter
  • Lombès Marc
  • Fagart Jérôme
  • Viengchareun Say
  • Martinerie Laetitia
  European Journal of Endocrinology, Oxford Univ. Press, 2024, 190 (4), pp.284-295. Abstract Objective Glucocorticoid resistance is a rare endocrine disease caused by variants of the NR3C1 gene encoding the glucocorticoid receptor (GR). We identified a novel heterozygous variant (GR R569Q) in a patient with uncommon reversible glucocorticoid resistance syndrome. Methods We performed ex vivo functional characterization of the variant in patient fibroblasts and in vitro through transient transfection in undifferentiated HEK 293T cells to assess transcriptional activity, affinity, and nuclear translocation. We studied the impact of the variant on the tertiary structure of the ligand-binding domain through 3D modeling. Results The patient presented initially with an adrenal adenoma with mild autonomous cortisol secretion and undetectable adrenocorticotropin hormone (ACTH) levels. Six months after surgery, biological investigations showed elevated cortisol and ACTH (urinary free cortisol 114 µg/24 h, ACTH 10.9 pmol/L) without clinical symptoms, evoking glucocorticoid resistance syndrome. Functional characterization of the GRR569Q showed decreased expression of target genes (in response to 100 nM cortisol: SGK1 control +97% vs patient +20%, P &lt; .0001) and impaired nuclear translocation in patient fibroblasts compared to control. Similar observations were made in transiently transfected cells, but higher cortisol concentrations overcame glucocorticoid resistance. GR R569Q showed lower ligand affinity (Kd GRWT: 1.73 nM vs GR R569Q: 4.61 nM). Tertiary structure modeling suggested a loss of hydrogen bonds between H3 and the H1–H3 loop. Conclusion This is the first description of a reversible glucocorticoid resistance syndrome with effective negative feedback on corticotroph cells regarding increased plasma cortisol concentrations due to the development of mild autonomous cortisol secretion. (10.1093/ejendo/lvae031)
  DOI : 10.1093/ejendo/lvae031
• Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells
  
  • Fedry Juliette
  • Silva Joana
  • Vanevic Mihajlo
  • Fronik Stanley
  • Mechulam Yves
  • Schmitt Emmanuelle
  • Des Georges Amédée
  • Faller William James
  • Förster Friedrich
  Molecular Cell, Cell Press, 2024, 84 (6), pp.1078-1089.e4. Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress. We find that polysomes are compressed, with up to 30% of ribosomes in helical polysomes or collided disomes, some of which are bound to the stress effector GCN1. The native collision interface extends beyond the in vitro-characterized 40S and includes the L1 stalk and eEF2, possibly contributing to translocation inhibition. The accumulation of unresolved tRNA-bound 80S and 60S and aberrant 40S configurations identifies potentially limiting steps in collision responses. Our work provides a global view of the translation machinery in response to persistent collisions and a framework for quantitative analysis of translation dynamics in situ. (10.1016/j.molcel.2024.01.015)
  DOI : 10.1016/j.molcel.2024.01.015
• Synthesis of photoactivatable molecules for the spatiotemporal control of cell motility with light
  
  • Lucas Morane
  • Joyeux Benjamin
  • Courtois Arthur
  • Romero Stéphane
  • Gautreau Alexis
  • Le Clainche Christophe
  • Nay Bastien
  , 2024.
• Inactivating negative regulators of cortical branched actin enhances persistence of single cell migration
  
  • Fokin Artem I
  • Boutillon Arthur
  • James John
  • Courtois Laura
  • Vacher Sophie
  • Simanov Gleb
  • Wang Yanan
  • Polesskaya Anna
  • Bièche Ivan
  • David Nicolas B
  • Gautreau Alexis M
  Journal of Cell Science, Company of Biologists, 2024, 137 (1), pp.jcs261332. The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knock-down (KD) or knock-out (KO), we combine the inactivation of the Arp2/3 inhibitory protein Arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit, CYFIP2, that all enhance the polymerization of cortical branched actin (CBA). Inactivation of the 3 CBA negative regulators increases migration persistence of human breast MCF10A cells, and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO, but not triple KD cells, the “super-migrator” phenotype was associated with a heterogenous down-regulation of vimentin expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells restored to a large extent normal persistence of single cell migration, suggesting that vimentin down-regulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression. (10.1242/jcs.261332)
  DOI : 10.1242/jcs.261332
• Post-translational modifications in prion diseases
  
  • Bizingre Chloé
  • Bianchi Clara
  • Baudry Anne
  • Alleaume-Butaux Aurélie
  • Schneider Benoit
  • Pietri Mathéa
  Frontiers in Molecular Neuroscience, Frontiers Media, 2024, 17, pp.1405415. More than 650 reversible and irreversible post-translational modifications (PTMs) of proteins have been listed so far. Canonical PTMs of proteins consist of the covalent addition of functional or chemical groups on target backbone amino-acids or the cleavage of the protein itself, giving rise to modified proteins with specific properties in terms of stability, solubility, cell distribution, activity, or interactions with other biomolecules. PTMs of protein contribute to cell homeostatic processes, enabling basal cell functions, allowing the cell to respond and adapt to variations of its environment, and globally maintaining the constancy of the milieu interieur (the body’s inner environment) to sustain human health. Abnormal protein PTMs are, however, associated with several disease states, such as cancers, metabolic disorders, or neurodegenerative diseases. Abnormal PTMs alter the functional properties of the protein or even cause a loss of protein function. One example of dramatic PTMs concerns the cellular prion protein (PrP C ), a GPI-anchored signaling molecule at the plasma membrane, whose irreversible post-translational conformational conversion (PTCC) into pathogenic prions (PrP Sc ) provokes neurodegeneration. PrP C PTCC into PrP Sc is an additional type of PTM that affects the tridimensional structure and physiological function of PrP C and generates a protein conformer with neurotoxic properties. PrP C PTCC into PrP Sc in neurons is the first step of a deleterious sequence of events at the root of a group of neurodegenerative disorders affecting both humans (Creutzfeldt–Jakob diseases for the most representative diseases) and animals (scrapie in sheep, bovine spongiform encephalopathy in cow, and chronic wasting disease in elk and deer). There are currently no therapies to block PrP C PTCC into PrP Sc and stop neurodegeneration in prion diseases. Here, we review known PrP C PTMs that influence PrP C conversion into PrP Sc . We summarized how PrP C PTCC into PrP Sc impacts the PrP C interactome at the plasma membrane and the downstream intracellular controlled protein effectors, whose abnormal activation or trafficking caused by altered PTMs promotes neurodegeneration. We discussed these effectors as candidate drug targets for prion diseases and possibly other neurodegenerative diseases. (10.3389/fnmol.2024.1405415)
  DOI : 10.3389/fnmol.2024.1405415