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Publications
Les 20 dernières publications
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Multiscale simulations reveal TDP-43 molecular-level interactions driving condensation
- Journal : Biophysical Journal
- 2023
- The RNA-binding protein TDP-43 is associated with mRNA processing and transport from the nucleus to the cytoplasm. TDP-43 localizes in the nucleus as well as accumulating in cytoplasmic condensates such as stress granules. Aggregation and formation of amyloid-like fibrils of cytoplasmic TDP-43 are hallmarks of numerous neurodegenerative diseases, most strikingly present in >90% of amyotrophic lateral sclerosis (ALS) patients. If excessive accumulation of cytoplasmic TDP-43 causes, or is caused by, neurodegeneration is presently not known. In this work, we use molecular dynamics simulations at multiple resolutions to explore TDP-43 self- and cross-interaction dynamics. A full-length molecular model of TDP-43, all 414 amino acids, was constructed from select structures of the protein functional domains (N-terminal domain, and two RNA recognition motifs, RRM1 and RRM2) and modeling of disordered connecting loops and the low complexity glycine-rich C-terminus domain. All-atom CHARMM36m simulations of single TDP-43 proteins served as guides to construct a coarse-grained Martini 3 model of TDP-43. The Martini model and a coarser implicit solvent C⍺ model, optimized for disordered proteins, were subsequently used to probe TDP-43 interactions; self-interactions from single-chain full-length TDP-43 simulations, cross-interactions from simulations with two proteins and simulations with assemblies of dozens to hundreds of proteins. Our findings illustrate the utility of different modeling scales for accessing TDP-43 molecular-level interactions and suggest that TDP-43 has numerous interaction preferences or patterns, exhibiting an overall strong, but dynamic, association and driving the formation of biomolecular condensates.
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AlphaFold2 Predicts Whether Proteins Interact Amidst Confounding Structural Compatibility
- Journal : Journal of Chemical Information and Modeling
- 2024
- Predicting whether two proteins physically interact is one of the holy grails of computational biology, galvanized by rapid advancements in deep learning. AlphaFold2, although not developed with this goal, is promising in this respect. Here, I test the prediction capability of AlphaFold2 on a very challenging data set, where proteins are structurally compatible, even when they do not interact. AlphaFold2 achieves high discrimination between interacting and non-interacting proteins, and the cases of misclassifications can either be rescued by revisiting the input sequences or can suggest false positives and negatives in the data set. AlphaFold2 is thus not impaired by the compatibility between protein structures and has the potential to be applied on a large scale.
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Dynamics of Protein–RNA Interfaces Using All-Atom Molecular Dynamics Simulations
- Journal : The Journal of Physical Chemistry B
- 2024
- Facing the current challenges posed by human health diseases requires the understanding of cell machinery at a molecular level. The interplay between proteins and RNA is key for any physiological phenomenon, as well protein–RNA interactions. To understand these interactions, many experimental techniques have been developed, spanning a very wide range of spatial and temporal resolutions. In particular, the knowledge of tridimensional structures of protein–RNA complexes provides structural, mechanical, and dynamical pieces of information essential to understand their functions. To get insights into the dynamics of protein–RNA complexes, we carried out all-atom molecular dynamics simulations in explicit solvent on nine different protein–RNA complexes with different functions and interface size by taking into account the bound and unbound forms. First, we characterized structural changes upon binding and, for the RNA part, the change in the puckering. Second, we extensively analyzed the interfaces, their dynamics and structural properties, and the structural waters involved in the binding, as well as the contacts mediated by them. Based on our analysis, the interfaces rearranged during the simulation time showing alternative and stable residue–residue contacts with respect to the experimental structure.
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Ultrastructure Expansion Microscopy applied to C. elegans embryos.
- Journal : MicroPubl Biol
- 2024
- Visualization of organelles using expansion microscopy has been previouslyapplied to Caenorhadbitis elegans adult gonads or worms. However, its applicationto embryos has remained a challenge due to the protective eggshell barrier. Here,by combining freeze-cracking and ultrastructure expansion microscopy (U-ExM), wedemonstrate a four-time isotropic expansion of C. elegans embryos. As an examplestructure, we chose the nuclear pore and demonstrate that we achieve sufficientresolution to distinguish them individually. Our work provides proof of principlefor U-ExM in C. elegans embryos, which will be applicable for imaging a widerange of cellular structures in this model system.
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Translation-dependent and -independent mRNA decay occur through mutually exclusive pathways defined by ribosome density during T cell activation.
- Journal : Genome Res
- 2024
- mRNA translation and decay are tightly interconnected processes both in thecontext of mRNA quality-control pathways and for the degradation of functionalmRNAs. Cotranslational mRNA degradation through codon usage, ribosome collisions,and the recruitment of specific proteins to ribosomes is an important determinantof mRNA turnover. However, the extent to which translation-dependent mRNA decay(TDD) and translation-independent mRNA decay (TID) pathways participate in thedegradation of mRNAs has not been studied yet. Here we describe a comprehensiveanalysis of basal and signal-induced TDD and TID in mouse primary CD4(+) T cells.Our results indicate that most cellular transcripts are decayed to some extent ina translation-dependent manner. Our analysis further identifies the length ofuntranslated regions, the density of ribosomes, and GC3 content as importantdeterminants of TDD magnitude. Consistently, all transcripts that undergo changesin ribosome density within their coding sequence upon T cell activation display acorresponding change in their TDD level. Moreover, we reveal a dynamic modulationin the relationship between GC3 content and TDD upon T cell activation, with areversal in the impact of GC3- and AU3-rich codons. Altogether, our data show astrong and dynamic interconnection between mRNA translation and decay inmammalian primary cells.
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Stable structures or PABP1 loading protects cellular and viral RNAs against ISG20-mediated decay.
- Journal : Life Sci Alliance
- 2024
- ISG20 is an IFN-induced 3'-5' RNA exonuclease that acts as a broad antiviralfactor. At present, the features that expose RNA to ISG20 remain unclear,although recent studies have pointed to the modulatory role of epitranscriptomicmodifications in the susceptibility of target RNAs to ISG20. These findings raisethe question as to how cellular RNAs, on which these modifications are abundant,cope with ISG20. To obtain an unbiased perspective on this topic, we used RNA-seqand biochemical assays to identify elements that regulate the behavior of RNAsagainst ISG20. RNA-seq analyses not only indicate a general preservation of thecell transcriptome, but they also highlight a small, but detectable, decrease inthe levels of histone mRNAs. Contrarily to all other cellular ones, histone mRNAsare non-polyadenylated and possess a short stem-loop at their 3' end, promptingus to examine the relationship between these features and ISG20 degradation. Theresults we have obtained indicate that poly(A)-binding protein loading on the RNA3' tail provides a primal protection against ISG20, easily explaining the overallprotection of cellular mRNAs observed by RNA-seq. Terminal stem-loop RNAstructures have been associated with ISG20 protection before. Here, were-examined this question and found that the balance between resistance andsusceptibility to ISG20 depends on their thermodynamic stability. These resultsshed new light on the complex interplay that regulates the susceptibility ofdifferent classes of viruses against ISG20.
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Metabolism-dependent secondary effect of anti-MAPK cancer therapy on DNA repair.
- Journal : NAR Cancer
- 2024
- Amino acid bioavailability impacts mRNA translation in a codon-dependent manner.Here, we report that the anti-cancer MAPK inhibitors (MAPKi) decrease theintracellular concentration of aspartate and glutamate in melanoma cells. Thiscoincides with the accumulation of ribosomes on codons corresponding to theseamino acids and triggers the translation-dependent degradation of mRNAs encodingaspartate- and glutamate-rich proteins, involved in DNA metabolism such as DNAreplication and repair. Consequently, cells that survive MAPKi degrade aspartateand glutamate likely to generate energy, which simultaneously decreases theirrequirement for amino acids due to the downregulation of aspartate- andglutamate-rich proteins involved in cell proliferation. Concomitantly, thedownregulation of aspartate- and glutamate-rich proteins involved in DNA repairincreases DNA damage loads. Thus, DNA repair defects, and therefore mutations,are at least in part a secondary effect of the metabolic adaptation of cellsexposed to MAPKi.
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Shaping the Innate Immune Response Through Post-Transcriptional Regulation of Gene Expression Mediated by RNA-Binding Proteins.
- Journal : Front Immunol
- 2021
- Innate immunity is the frontline of defense against infections and tissue damage.It is a fast and semi-specific response involving a myriad of processes essentialfor protecting the organism. These reactions promote the clearance of danger byactivating, among others, an inflammatory response, the complement cascade and byrecruiting the adaptive immunity. Any disequilibrium in this functional balancecan lead to either inflammation-mediated tissue damage or defense inefficiency. Adynamic and coordinated gene expression program lies at the heart of the innateimmune response. This expression program varies depending on the cell-type andthe specific danger signal encountered by the cell and involves multiple layersof regulation. While these are achieved mainly via transcriptional control ofgene expression, numerous post-transcriptional regulatory pathways involvingRNA-binding proteins (RBPs) and other effectors play a critical role in itsfine-tuning. Alternative splicing, translational control and mRNA stability havebeen shown to be tightly regulated during the innate immune response andparticipate in modulating gene expression in a global or gene specific manner.More recently, microRNAs assisting RBPs and post-transcriptional modification ofRNA bases are also emerging as essential players of the innate immune process. Inthis review, we highlight the numerous roles played by specific RNA-bindingeffectors in mediating post-transcriptional control of gene expression to shapeinnate immunity.
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The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses.
- Journal : EMBO Rep
- 2022
- Genome-wide screens are powerful approaches to unravel regulators of viralinfections. Here, a CRISPR screen identifies the RNA helicase DDX42 as anintrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increasesHIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42overexpression inhibits HIV-1 infection, whereas expression of adominant-negative mutant increases infection. Importantly, DDX42 also restrictsLINE-1 retrotransposition and infection with other retroviruses andpositive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 doesnot impact the replication of several negative-strand RNA viruses, arguingagainst an unspecific effect on target cells, which is confirmed by RNA-seqanalysis. Proximity ligation assays show DDX42 in the vicinity of viral elements,and cross-linking RNA immunoprecipitation confirms a specific interaction ofDDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibitsHIV-1 reverse transcription in vitro. Together, our data strongly suggest adirect mode of action of DDX42 on viral ribonucleoprotein complexes. Our resultsidentify DDX42 as an intrinsic viral inhibitor, opening new perspectives totarget the life cycle of numerous RNA viruses.
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RSL24D1 sustains steady-state ribosome biogenesis and pluripotency translational programs in embryonic stem cells.
- Journal : Nat Commun
- 2023
- Embryonic stem cell (ESC) fate decisions are regulated by a complex circuitrythat coordinates gene expression at multiple levels from chromatin to mRNAprocessing. Recently, ribosome biogenesis and translation have emerged as keypathways that efficiently control stem cell homeostasis, yet the underlyingmolecular mechanisms remain largely unknown. Here, we identified RSL24D1 ashighly expressed in both mouse and human pluripotent stem cells. RSL24D1 isassociated with nuclear pre-ribosomes and is required for the biogenesis of 60Ssubunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairsglobal translation, particularly of key pluripotency factors and of componentsfrom the Polycomb Repressive Complex 2 (PRC2). While having a moderate impact ondifferentiation, RSL24D1 depletion significantly alters ESC self-renewal andlineage commitment choices. Altogether, these results demonstrate thatRSL24D1-dependant ribosome biogenesis is both required to sustain the expressionof pluripotent transcriptional programs and to silence PRC2-regulateddevelopmental programs, which concertedly dictate ESC homeostasis.
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Severe COVID-19 patients have impaired plasmacytoid dendritic cell-mediated control of SARS-CoV-2.
- Journal : Nat Commun
- 2023
- Type I and III interferons (IFN-I/λ) are important antiviral mediators againstSARS-CoV-2 infection. Here, we demonstrate that plasmacytoid dendritic cells(pDC) are the predominant IFN-I/λ source following their sensing ofSARS-CoV-2-infected cells. Mechanistically, this short-range sensing by pDCsrequires sustained integrin-mediated cell adhesion with infected cells. In turn,pDCs restrict viral spread by an IFN-I/λ response directed towardSARS-CoV-2-infected cells. This specialized function enables pDCs to efficientlyturn-off viral replication, likely via a local response at the contact site withinfected cells. By exploring the pDC response in SARS-CoV-2 patients, we furtherdemonstrate that pDC responsiveness inversely correlates with the severity of thedisease. The pDC response is particularly impaired in severe COVID-19 patients.Overall, we propose that pDC activation is essential to controlSARS-CoV-2-infection. Failure to develop this response could be important tounderstand severe cases of COVID-19.
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SIN-3 transcriptional coregulator maintains mitochondrial homeostasis and polyamine flux
- Journal : iScience
- 2024
- Summary Mitochondrial function relies on the coordinated transcription of mitochondrial and nuclear genomes to assemble respiratory chain complexes. Across species, the SIN3 coregulator influences mitochondrial functions, but how its loss impacts mitochondrial homeostasis and metabolism in the context of a whole organism is unknown. Exploring this link is important because SIN3 haploinsufficiency causes intellectual disability/autism syndromes and SIN3 plays a role in tumor biology. Here we show that loss of C. elegans SIN-3 results in transcriptional deregulation of mitochondrial- and nuclear-encoded mitochondrial genes, potentially leading to mito-nuclear imbalance. Consistent with impaired mitochondrial function, sin-3 mutants show extensive mitochondrial fragmentation by transmission electron microscopy (TEM) and in vivo imaging, and altered oxygen consumption. Metabolomic analysis of sin-3 mutant animals revealed a mitochondria stress signature and deregulation of methionine flux, resulting in decreased S-adenosyl methionine (SAM) and increased polyamine levels. Our results identify SIN3 as a key regulator of mitochondrial dynamics and metabolic flux, with important implications for human pathologies.
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Yeast cell responses and survival during periodic osmotic stress are controlled by glucose availability
- Journal : Elife
- 2024
- Natural environments of living organisms are often dynamic and multifactorial, with multiple parameters fluctuating over time. To better understand how cells respond to dynamically interacting factors, we quantified the effects of dual fluctuations of osmotic stress and glucose deprivation on yeast cells using microfluidics and time-lapse microscopy. Strikingly, we observed that cell proliferation, survival, and signaling depend on the phasing of the two periodic stresses. Cells divided faster, survived longer, and showed decreased transcriptional response when fluctuations of hyperosmotic stress and glucose deprivation occurred in phase than when the two stresses occurred alternatively. Therefore, glucose availability regulates yeast responses to dynamic osmotic stress, showcasing the key role of metabolic fluctuations in cellular responses to dynamic stress. We also found that mutants with impaired osmotic stress response were better adapted to alternating stresses than wild-type cells, showing that genetic mechanisms of adaptation to a persistent stress factor can be detrimental under dynamically interacting conditions.
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RNAP II antagonizes mitotic chromatin folding and chromosome segregation by condensin.
- Journal : Cell Rep
- 2024
- Condensin shapes mitotic chromosomes by folding chromatin into loops, but whetherit does so by DNA-loop extrusion remains speculative. Although loop-extrudingcohesin is stalled by transcription, the impact of transcription on condensin,which is enriched at highly expressed genes in many species, remains unclear.Using degrons of Rpb1 or the torpedo nuclease Dhp1(XRN2) to either deplete ordisplace RNAPII on chromatin in fission yeast metaphase cells, we show thatRNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cisand hinders its ability to fold mitotic chromatin and to support chromosomesegregation, consistent with the stalling of a loop extruder. Transcriptiontermination by Dhp1 limits such a hindrance. Our results shed light on theintegrated functioning of condensin, and we argue that a tight control oftranscription underlies mitotic chromosome assembly by loop-extruding condensin.
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Discriminating physiological from non-physiological interfaces in structures of protein complexes: a community-wide study
- Journal : Proteomics
- 2023
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Expulsion mechanism of the substrate-translocating subunit in ECF transporters
- Journal : Nature Communications
- 2023
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Transmembrane dimers of type 1 receptors sample alternate configurations: MD simulations using coarse grain Martini 3 versus AlphaFold2 Multimer
- Journal : Structure
- 2023
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An implementation of the Martini coarse-grained force field in OpenMM
- Journal : Biophysical Journal
- 2023
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Mono- and Intralink Filter (Mi-Filter) To Reduce False Identifications in Cross-Linking Mass Spectrometry Data.
- Journal : Anal Chem
- 2022
- Cross-linking mass spectrometry (XL-MS) has become an indispensable tool for theemerging field of systems structural biology over the recent years. However, theconfidence in individual protein-protein interactions (PPIs) depends on thecorrect assessment of individual inter-protein cross-links. In this article, wedescribe a mono- and intralink filter (mi-filter) that is applicable to any kindof cross-linking data and workflow. It stipulates that only proteins for which atleast one monolink or intra-protein cross-link has been identified within a givendata set are considered for an inter-protein cross-link and therefore participatein a PPI. We show that this simple and intuitive filter has a dramatic effect ondifferent types of cross-linking data ranging from individual protein complexesover medium-complexity affinity enrichments to proteome-wide cell lysates andsignificantly reduces the number of false-positive identifications forinter-protein links in all these types of XL-MS data.
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Coevolution-Guided Mapping of the Type VI Secretion Membrane Complex-Baseplate Interface.
- Journal : J Mol Biol
- 2023
- The type VI secretion system (T6SS) is a multiprotein weapon evolved byGram-negative bacteria to deliver effectors into eukaryotic cells or bacterialrivals. The T6SS uses a contractile mechanism to propel an effector-loaded needleinto its target. The contractile tail is built on an assembly platform, thebaseplate, which is anchored to a membrane complex. Baseplate-membrane complexinteractions are mainly mediated by contacts between the C-terminal domain of theTssK baseplate component and the cytoplasmic domain of the TssL inner membraneprotein. Currently, the structural details of this interaction are unknown due tothe marginal stability of the TssK-TssL complex. Here we conducted a mutagenesisstudy based on putative TssK-TssL contact pairs identified by co-evolutionanalyses. We then evaluated the impact of these mutations on T6SS activity,TssK-TssL interaction and sheath assembly and dynamics in enteroaggregativeEscherichia coli. Finally, we probed the TssK-TssL interface by disulfidecross-linking, allowing to propose a model for the baseplate-membrane complexinterface.
Link to PubMed entry