Publications

2023

• The YTHDF1-3 proteins are bidirectionally influenced by the codon content of their mRNA targets
  
  • Moch Clara
  • Zou Limei
  • Pythoud Nicolas
  • Fillon Emilie
  • Bourgeois Gabrielle
  • Graille Marc
  • Carapito Christine
  • Chapat Clement
  , 2023. N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNAs and plays critical roles in a broad variety of biological processes. Recognition of m6A by the YTHDF1-3 proteins results in the alteration of the translation efficiency and stability of methylated mRNAs, although their mode of action is still matter of debates. To decode the molecular basis of YTHDF1-3 action in human cells, we performed an unbiased proteomic screen of their full spectrum of interacting proteins using BioID (proximity-dependent biotin identification). Our systematic BioID mapping revealed that each YTHDF protein is a dynamic hub that associates with both mRNA silencing machineries and the translation apparatus. Based on this, we identified a direct interaction between YTHDF2 and the ribosomal protein RACK1, and found that the silencing activity of YTHDF2 is bidirectionally modulated by the codon content of its targeted mRNAs. Using a tethering reporter system that recapitulates this phenomenon, we confirmed that the three YTHDF proteins selectively repress mRNAs enriched in optimal codons, while they activate those enriched in non-optimal codons. Altogether, these results have important implications for understanding the underlying multiplicity of YTHDF1-3 and could reconcile seemingly contradictory data. (10.1101/2023.11.20.565808)
  DOI : 10.1101/2023.11.20.565808
• Machine learning to understand and engineer the structural kinome
  
  • Reveguk Ivan
  , 2023. Protein kinases (PKs) comprise one of the most ancient and ubiquitous enzyme groups deeply embedded in a cell's molecular machinery. Changing their targets' conformation via phosphate transfer, PKs themselves cycle between active and inactive states. Any misbalance between them can lead to harmful diseases, including cancers. The DFG motif, situated within the activation loop (AL), displays conformational variability in the less constrained inactive state: a property that small molecule inhibitors often exploit. Namely, there are two major DFG motif orientations, known as the DFG-in and DFG-out. The latter precludes substrate binding and is typically associated with higher inhibitor selectivity. Despite being pivotal to drug design efforts, little is known regarding which features shape AL conformational landscape. This work constitutes a systematic attempt to uncover them via careful data curation and mining. Over its course, we created the largest structural kinome assembly to date, encompassing nearly ten thousand annotated PK domains. Clustering these domains enabled semi-supervised labeling of the DFG motif conformations. These labels served as input to our interpretable machine-learning (ML) pipeline incorporating decision tree-based ensembles and a model-agnostic feature selection algorithm. The obtained classifiers accurately predicted the DFG conformations and active/inactive states and relied on readily interpretable structural hallmarks. We used the obtained annotations and ML models' predictions to characterize sequence elements likely responsible for shifting the AL inactive state's conformational balance, or "discriminative" positions. To uncover these, we created several sequence-based datasets, each having a different level of conformational propensity attributed to a sequence. We used the same ML pipeline and phylogenetic analysis to show that a clear DFG conformational propensity is likely privileged to a group of closely related receptor tyrosine protein kinases. The discovered discriminating positions overlapped with the existing literature and mutagenesis studies and may provide a foundation for future experimental efforts, including computational protein design applications. Finally, the developed methodology enables automating the annotation of the structural kinome. Generalizable towards problems of similar nature, it may increase the efficiency and transparency of data mining in structural biology.
• Involvement of eIF2 in cardiomyocytes response to endoplasmic reticulum stress
  
  • Cardenal Peralta Cristina
  , 2023. Over the last decade, endoplasmic reticulum (ER) stress has emerged as an important mechanism involved in the development of cardiac diseases including ischemia, dilated cardiomyopathy and heart failure. ER stress triggers the Unfolded Protein Response (UPR) initiated by the phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2). It is considered that a mild-to-moderate induction of ER stress triggers a restorative autophagy-based response whereas severe or chronic ER stress promotes an apoptotic response which contributes to the development of the cardiac disease. Therefore, cardiac therapy based on ER stress modulation is emerging as a promising new approach to promote beneficial adaptations and avoid apoptosis.Our INSERM's collaborator showed that sirtuin 1 (SIRT1), an NAD+-dependent lysine deacetylase, protects cardiomyocytes from ER stress-induced apoptosis by specifically attenuating activation of the eIF2α phosphorylation pathway and promoting autophagy. They also identified acetylation of two lysines of eIF2α, K141 and K143 and demonstrated that SIRT1 deacetylates these lysines to protect the heart from severe ER stress.No information is available on the consequences of eIF2α acetylation on its structural organization and its function. The aim of this PhD project is to decipher the molecular and structural basis of the SIRT1:eIF2α interaction. During this PhD, we were able to successfully purify an acetylated version eIF2α using an orthogonal system derived from the archaeal pyrrolysine system. Using this system, an acetyl-lysine was introduced on the place of a STOP codon. After extensive optimization, we were able to use this protein to test SIRT1 catalytic activity using western blotting. We also produced an acetylated version of the protein which carried an S52D mutation, mimicking phosphorylation, and were able to also obtain catalytic rates using this version of the protein.We also characterized SIRT1’s catalytic activity using mimic peptides of 143 lysine acetylation in eIF2α. The use of peptides in the literature is controversial because most of them are modified with a hydrophobic moiety that can affect SIRT1 affinity for its substrate. In our approach, we use non-modified peptides, closer to the native substrate.During this thesis, we were able to obtain a crystallographic structure of a mimic of eIF2α acetylation. Finally, we initiated crystallization trials with SIRT1:peptide substrates and SIRT1:eIF2α.
• Codependencies of mTORC1 signaling and endolysosomal actin structures
  
  • Priya Amulya
  • Antoine-Bally Sandra
  • Macé Anne-Sophie
  • Monteiro Pedro
  • Sabatet Valentin
  • Remy David
  • Dingli Florent
  • Loew Damarys
  • Demetriades Constantinos
  • Gautreau Alexis
  • Chavrier Philippe
  Science Advances, American Association for the Advancement of Science (AAAS), 2023, 9 (37), pp.eadd9084. The mechanistic target of rapamycin complex 1 (mTORC1) is part of the amino acid sensing machinery that becomes activated on the endolysosomal surface in response to nutrient cues. Branched actin generated by WASH and Arp2/3 complexes defines endolysosomal microdomains. Here, we find mTORC1 components in close proximity to endolysosomal actin microdomains. We investigated for interactors of the mTORC1 lysosomal tether, RAGC, by proteomics and identified multiple actin filament capping proteins and their modulators. Perturbation of RAGC function affected the size of endolysosomal actin, consistent with a regulation of actin filament capping by RAGC. Reciprocally, the pharmacological inhibition of actin polymerization or alteration of endolysosomal actin obtained upon silencing of WASH or Arp2/3 complexes impaired mTORC1 activity. Mechanistically, we show that actin is required for proper association of RAGC and mTOR with endolysosomes. This study reveals an unprecedented interplay between actin and mTORC1 signaling on the endolysosomal system. (10.1126/sciadv.add9084)
  DOI : 10.1126/sciadv.add9084
• Implementation of an orthogonal cysteine translation initiation system in E. coli
  
  • Paupelin-Vaucelle, Humbeline
  • Boschiero Claire
  • Lazennec-Schurdevin Christine
  • Mechulam Yves
  • Schmitt Emmanuelle
  • Marliere Philippe
  • Pezo Valérie
  , 2023.
• PLP-Dependent Enzyme Methionine γ-Lyase: Insights into the Michaelis Complex from Molecular Dynamics and Free Energy Simulations
  
  • Chen Xingyu
  • Ferchaud Nathan
  • Briozzo Pierre
  • Machover David
  • Simonson Thomas
  Biochemistry, American Chemical Society, 2023, 62 (18), pp.2791-2801. Methionine γ-lyase (MGL) breaks down methionine, with the help of its cofactor pyridoxal-5′-phosphate (PLP), or vitamin B6. Methionine depletion is damaging for cancer cells but not normal cells, so MGL is of interest as a therapeutic protein. To increase our understanding and help engineer improved activity, we focused on the reactive, Michaelis complex between MGL, covalently bound PLP, and substrate Met. is not amenable to crystallography, as it proceeds to products. Experimental activity measurements helped exclude a mechanism that would bypass. We then used molecular dynamics and alchemical free energy simulations to elucidate its structure and dynamics. We showed that the PLP phosphate has a pK a strongly downshifted by the protein, whether Met is present or not. Met binding affects the structure surrounding the reactive atoms. With Met, the Schiff base linkage between PLP and a nearby lysine shifts from a zwitterionic, keto form to a neutral, enol form that makes it easier for Met to approach its labile, target atom. The Met ligand also stabilizes the correct orientation of the Schiff base, more strongly than in simulations without Met, and in agreement with structures in the Protein Data Bank, where the Schiff base orientation correlates with the presence or absence of a co-bound anion or substrate analogue in the active site. Overall, the Met ligand helps organize the active site for the enzyme reaction by reducing fluctuations and shifting protonation states and conformational populations. (10.1021/acs.biochem.3c00355)
  DOI : 10.1021/acs.biochem.3c00355
• Redesigning methionyl‐tRNA synthetase for β‐methionine activity with adaptive landscape flattening and experiments
  
  • Opuu Vaitea
  • Nigro Giuliano
  • Lazennec-Schurdevin Christine
  • Mechulam Yves
  • Schmitt Emmanuelle
  • Simonson Thomas
  Protein Science, Wiley, 2023, 32 (9). Abstract Amino acids (AAs) with a noncanonical backbone would be a valuable tool for protein engineering, enabling new structural motifs and building blocks. To incorporate them into an expanded genetic code, the first, key step is to obtain an appropriate aminoacyl‐tRNA synthetase. Currently, directed evolution is not available to optimize AAs with noncanonical backbones, since an appropriate selective pressure has not been discovered. Computational protein design (CPD) is an alternative. We used a new CPD method to redesign MetRS and increase its activity towards β‐Met, which has an extra backbone methylene. The new method considered a few active site positions for design and used a Monte Carlo exploration of the corresponding sequence space. During the exploration, a bias energy was adaptively learned, such that the free energy landscape of the apo enzyme was flattened. Enzyme variants could then be sampled, in the presence of the ligand and the bias energy, according to their β‐Met binding affinities. Eighteen predicted variants were chosen for experimental testing; 10 exhibited detectable activity for β‐Met adenylation. Top predicted hits were characterized experimentally in detail. Dissociation constants, catalytic rates, and Michaelis constants for both α‐Met and β‐Met were measured. The best mutant retained a preference for α‐Met over β‐Met; however, the preference was reduced, compared to the wildtype, by a factor of 29. For this mutant, high resolution crystal structures were obtained in complex with both α‐Met and β‐Met, indicating that the predicted, active conformation of β‐Met in the active site was retained. (10.1002/pro.4738)
  DOI : 10.1002/pro.4738
• Binding of human Cdc123 to eIF2γ
  
  • Cardenal Peralta Cristina
  • Vandroux Paul
  • Neumann-Arnold Lea
  • Panvert Michel
  • Fagart Jérôme
  • Seufert Wolfgang
  • Mechulam Yves
  • Schmitt Emmanuelle
  Journal of Structural Biology, Elsevier, 2023, 215 (3), pp.108006. Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a member of the ATP grasp family that binds the γ subunit of eIF2. Notably, some mutations related to MEHMO syndrome, an X-linked intellectual disability, affect Cdc123-mediated eIF2 assembly. The mechanism of action of Cdc123 is unclear and structural information for the human protein is awaited. Here, the crystallographic structure of human Cdc123 (Hs-Cdc123) bound to domain 3 of human eIF2γ (Hs-eIF2γD3) was determined. The structure shows that the domain 3 of eIF2γ is bound to domain 1 of Cdc123. In addition, the long C-terminal region of Hs-Cdc123 provides a link between the ATP and Hs-eIF2γD3 binding sites. A thermal shift assay shows that ATP is tightly bound to Cdc123 whereas the affinity of ADP is much smaller. Yeast cell viability experiments, western blot analysis and two-hybrid assays show that ATP is important for the function of Hs-Cdc123 in eIF2 assembly. These data and recent findings allow us to propose a refined model to explain the mechanism of action of Cdc123 in eIF2 assembly. (10.1016/j.jsb.2023.108006)
  DOI : 10.1016/j.jsb.2023.108006
• Vinculin-Arp2/3 interaction antagonises branched actin to control single and collective cell behaviours
  
  • James John
  , 2023. Development, growth and maintenance of tissues are emergent properties arising from individual cell behaviour. Cell behaviour is finely tuned by a multitude of regulatory pathways in response to stimuli received from their microenvironment. Physical forces are sensed at cell-substrate contacts called focal adhesions (FAs) and cell-cell contacts called adherens junctions (AJs) which connect the exterior of a cell to its actin cytoskeleton. In response to force sensing, the actin cytoskeleton is remodelled to regulate complex cell behaviours such as proliferation, migration and cell-junction maintenance that are under the control of branched actin. During cancer progression, these three processes are deregulated. Vinculin (VCL), described as a tumour suppressor, is a structural and mechanotransductory protein present in both FAs and AJs. In addition to reinforcing the link between the actin cytoskeleton and adhesive structures, VCL is likely to plays a second regulatory role on the actin cytoskeleton by interacting with the branched actin nucleator Arp2/3. The goal of this project was to determine the effects of VCL on branched actin, and ultimately cell behaviour. To this end, we used CRISPR-Cas9 genome editing techniques to perturb this interaction.We began studying the effects of the interaction on individual cell behaviour by expressing a VCL 811-881 peptide in Parental MCF10A cells. We found that the peptide binds the canonical Arp2/3 complex, and cells expressing the peptide are able to migrate more persistently, spread over a larger area, make larger lamellipodia and continue to proliferate at high cell densities. All these phenotypes indicate that Arp2/3 activity is increased in these cells. VCL knockout (VCL-/-) cell lines and a mutant cell line where VCL cannot bind Arp2/3 (VCL P878A-KI) both behave similarly. TIRF-SIM imaging revealed that the actin network assembly rate was increased in VCL-/-, VCL P878A-KI and VCL 811-881 expressing cells compared to Parental MCF10A. Together, this demonstrates that the function of the VCL-Arp2/3 interaction is to antagonize generation of branched actin networks in the lamellipodium, and that the VCL 811-881 peptide acts as a dominant negative of VCL function.To understand the role of the VCL-Arp2/3 interaction on collective cell behaviour, we first studied AJs which not only hold cells together but also allow them to pass on mechanical signals. We found that the VCL controls Arp2/3 recruitment to AJs and cell-cell junction stability. Once AJs were mature, Parental MCF10A cells were able to organise long-range transcellular actin networks coordinated across multiple cells. During hypotonic unjamming, these cells migrate collectively as domains constrained by the long-range actin network. VCL P878A cells are not constrained by any long-range transcellular actin network and migrate more collectively while VCL-/- cells develop a very short-range actin network leading to reduced collective migration. Our results indicate that Arp2/3 activity has to be finely regulated at AJs by VCL to form this long range network and regulate collective migration.Thus, we have established as a novel role for VCL in regulating the actin cytoskeleton through a direct interaction that antagonizes Arp2/3. Perturbation of this interaction leads to several phenotypes characteristic of cancer cells – increased proliferation, increased persistence of migration, perturbation of cell junctions and misregulation of collective migration. Since Arp2/3 activity is known to be upregulated in several cancer types, our results provide a potential mechanism for vinculin’s role as a tumour suppressor.
• Implementation of an orthogonal cysteine translation initiation system in E. coli
  
  • Paupelin-Vaucelle, Humbeline
  • Boschiero Claire
  • Lazennec-Schurdevin Christine
  • Mechulam Yves
  • Schmitt Emmanuelle
  • Marliere Philippe
  • Pezo Valérie
  , 2023.
• Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea
  
  • Grünberger Felix
  • Jüttner Michael
  • Knüppel Robert
  • Ferreira-Cerca Sébastien
  • Grohmann Dina
  RNA, Cold Spring Harbor Laboratory Press, 2023, 29 (8), pp.1255-1273. Ribosomal RNA (rRNA) maturation in archaea is a complex multistep process that requires well-defined endo- and exoribonuclease activities to generate fully mature linear rRNAs. However, technical challenges prevented detailed mapping of rRNA processing steps and a systematic analysis of rRNA maturation pathways across the tree of life. In this study, we used long-read (PCR)-cDNA and direct RNA nanopore-based sequencing to study rRNA maturation in three archaeal model organisms, namely the Euryarchaea Haloferax volcanii and Pyrococcus furiosus and the Crenarchaeon Sulfolobus acidocaldarius . Compared to standard short-read protocols, nanopore sequencing facilitates simultaneous readout of 5′- and 3′-positions, which is required for the classification of rRNA processing intermediates. More specifically, we (i) accurately detect and describe rRNA maturation stages by analysis of terminal read positions of cDNA reads and thereupon (ii) explore the stage-dependent installation of the KsgA-mediated dimethylations in H. volcanii using base-calling and signal characteristics of direct RNA reads. Due to the single-molecule sequencing capacity of nanopore sequencing, we could detect hitherto unknown intermediates with high confidence, revealing details about the maturation of archaea-specific circular rRNA intermediates. Taken together, our study delineates common principles and unique features of rRNA processing in euryarchaeal and crenarchaeal representatives, thereby significantly expanding our understanding of rRNA maturation pathways in archaea. (10.1261/rna.079636.123)
  DOI : 10.1261/rna.079636.123
• ERH : a plug‐and‐play protein important for gene silencing and cell cycle progression
  
  • Graille Marc
  FEBS Journal, Wiley, 2023, 290 (3), pp.688-691. In metazoans, most proteins have pleiotropic cellular functions and have the ability to interact with several factors to accomplish these different functions. This is the case of eukaryotic ERH proteins, a family of homodimeric proteins involved in DNA replication and cell cycle control as well as in gene silencing by contributing either to the biogenesis of small interference RNAs (miRNAs, piRNAs) or to the recruitment of RNA decay machineries. Very recently, several crystal structures describing complexes formed by eukaryotic ERH proteins and several small peptides from various partners have highlighted the existence of different binding sites on the surface of ERH proteins. In this issue of The FEBS Journal , Wang et al. present the crystal structure of the complex formed between the human ERH protein and a short peptide of the CIZ1 protein, one of its partners. Altogether, this information will be particularly important for future studies aimed at dissecting the different biological functions governed by this family of highly conserved proteins. Comment on: https://doi.org/10.1111/febs.16611 (10.1111/febs.16669)
  DOI : 10.1111/febs.16669
• Enzyme redesign and genetic code expansion
  
  • Opuu Vaitea
  • Simonson Thomas
  Protein Engineering, Design and Selection, Oxford University Press (OUP), 2023, 36. Abstract Enzyme design is an important application of computational protein design (CPD). It can benefit enormously from the additional chemistries provided by noncanonical amino acids (ncAAs). These can be incorporated into an ‘expanded’ genetic code, and introduced in vivo into target proteins. The key step for genetic code expansion is to engineer an aminoacyl-transfer RNA (tRNA) synthetase (aaRS) and an associated tRNA that handles the ncAA. Experimental directed evolution has been successfully used to engineer aaRSs and incorporate over 200 ncAAs into expanded codes. But directed evolution has severe limits, and is not yet applicable to noncanonical AA backbones. CPD can help address several of its limitations, and has begun to be applied to this problem. We review efforts to redesign aaRSs, studies that designed new proteins and functionalities with the help of ncAAs, and some of the method developments that have been used, such as adaptive landscape flattening Monte Carlo, which allows an enzyme to be redesigned with substrate or transition state binding as the design target. (10.1093/protein/gzad017)
  DOI : 10.1093/protein/gzad017
• Structural insights into the evolution of late steps of translation initiation in the three domains of life
  
  • Kazan Ramy
  • Bourgeois Gabrielle
  • Lazennec-Schurdevin Christine
  • Coureux Pierre-Damien
  • Mechulam Yves
  • Schmitt Emmanuelle
  Biochimie, Elsevier, 2023, pp.S0300-9084(23)00030-5. In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. These two factors are also orthologous to the bacterial IF2 and IF1 proteins, respectively. Recent cryo-EM studies showed how e/aIF5B and e/aIF1A cooperate on the small ribosomal subunit to favor the binding of the large ribosomal subunit and the formation of a ribosome competent for elongation. In this review, pioneering studies and recent biochemical and structural results providing new insights into the role of a/eIF5B in archaea and eukaryotes will be presented. Recent structures will also be compared to orthologous bacterial initiation complexes to highlight domain-specific features and the evolution of initiation mechanisms. (10.1016/j.biochi.2023.02.002)
  DOI : 10.1016/j.biochi.2023.02.002
• Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius
  
  • Baes Rani
  • Grünberger Felix
  • Pyr Dit Ruys Sébastien
  • Couturier Mohea
  • de Keulenaer Sarah
  • Skevin Sonja
  • van Nieuwerburgh Filip
  • Vertommen Didier
  • Grohmann Dina
  • Ferreira-Cerca Sébastien
  • Peeters Eveline
  mBio, American Society for Microbiology, 2023. High-temperature stress is critical for all organisms and induces a profound cellular response. For Crenarchaeota, little information is available on how heat shock affects cellular processes and on how this response is regulated. We set out to study heat shock response in the thermoacidophilic model crenarchaeon Sulfolobus acidocaldarius, which thrives in volcanic hot springs and has an optimal growth temperature of 75°C. Pulse-labeling experiments demonstrated that a temperature shift to 86°C induces a drastic reduction of the transcriptional and translational activity, but that RNA and protein neosynthesis still occurs. By combining RNA sequencing and mass spectrometry, an integrated mapping of the transcriptome and proteome was performed. This revealed that heat shock causes an immediate change in the gene expression profile, with RNA levels of half of the genes being affected, followed by a more subtle reprogramming of the protein landscape. Functional enrichment analysis indicated that nearly all cellular processes are affected by heat shock. A limited correlation was observed in the differential expression on the RNA and protein level, suggesting a prevalence of post-transcriptional and post-translational regulation. Furthermore, promoter sequence analysis of heat shock regulon genes demonstrated the conservation of strong transcription initiation elements for highly induced genes, but an absence of a conserved protein-binding motif. It is, therefore, hypothesized that histone-lacking archaea such as Sulfolobales use an evolutionarily ancient regulatory mechanism that relies on temperature-responsive changes in DNA organization and compaction induced by the action of nucleoid-associated proteins, as well as on enhanced recruitment of initiation factors. (10.1128/mbio.03593-22)
  DOI : 10.1128/mbio.03593-22
• Best Practices of Using AI-Based Models in Crystallography and Their Impact in Structural Biology
  
  • Graille Marc
  • Sacquin-Mora Sophie
  • Taly Antoine
  Journal of Chemical Information and Modeling, American Chemical Society, 2023. The recent breakthrough made in the field of 3D structure prediction by artificial intelligence softwares such as initially AlphaFold2 1 (AF2) and RosettaFold 2 (RF) and more recently large Language Models 3 (LLM), has revolutionized the field of structural biology in particular but also biology as a whole. These models have clearly generated a great enthusiasm within the scientific community and different applications of these 3D predictions are regularly described in scientific articles demonstrating the impact of these high quality models. Despite the acknowledged high accuracy of these models in general, it seems important to make users of these models aware of the wealth of information they offer and to encourage them to make the best use of them. Here, we focus on the impact of these models in a specific application by structural biologists using X-ray crystallography. We propose guidelines to prepare models to be used for molecular replacement trials to solve the phase problem. We also encourage colleagues to share as much detail as possible about how they use these models in their research, where the models did not yield correct molecular replacement solutions, and how these predictions fit with their experimental 3D structure. We feel this is important to improve the pipelines using these models and also to get feedback on their overall quality. (10.1021/acs.jcim.3c00381)
  DOI : 10.1021/acs.jcim.3c00381
• Computational protein design repurposed to explore enzyme vitality and help predict antibiotic resistance
  
  • Michael Eleni
  • Saint-Jalme Rémy
  • Mignon David
  • Simonson Thomas
  Frontiers in Molecular Biosciences, Frontiers Media, 2023, 9, pp.905588. In response to antibiotics that inhibit a bacterial enzyme, resistance mutations inevitably arise. Predicting them ahead of time would aid target selection and drug design. The simplest resistance mechanism would be to reduce antibiotic binding without sacrificing too much substrate binding. The property that reflects this is the enzyme "vitality", defined here as the difference between the inhibitor and substrate binding free energies. To predict such mutations, we borrow methodology from computational protein design. We use a Monte Carlo exploration of mutation space and vitality changes, allowing us to rank thousands of mutations and identify ones that might provide resistance through the simple mechanism considered. As an illustration, we chose dihydrofolate reductase, an essential enzyme targeted by several antibiotics. We simulated its complexes with the inhibitor trimethoprim and the substrate dihydrofolate. 20 active site positions were mutated, or "redesigned" individually, then in pairs or quartets. We computed the resulting binding free energy and vitality changes. Out of seven known resistance mutations involving active site positions, five were correctly recovered. Ten positions exhibited mutations with significant predicted vitality gains. Direct couplings between designed positions were predicted to be small, which reduces the combinatorial complexity of the mutation space to be explored. It also suggests that over the course of evolution, resistance mutations involving several positions do not need the underlying point mutations to arise all at once: they can appear and become fixed one after the other. (10.3389/fmolb.2022.905588)
  DOI : 10.3389/fmolb.2022.905588
• PPP2R1A regulates migration persistence through the NHSL1-containing WAVE Shell Complex
  
  • Wang Yanan
  • Chiappetta Giovanni
  • Guérois Raphaël
  • Liu Yijun
  • Romero Stéphane
  • Boesch Daniel
  • Krause Matthias
  • Dessalles Claire
  • Babataheri Avin
  • Barakat Abdul
  • Chen Baoyu
  • Vinh Joelle
  • Polesskaya Anna
  • Gautreau Alexis
  Nature Communications, Nature Publishing Group, 2023, 14 (1), pp.3541. The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Here, we identify PPP2R1A by proteomics as a protein differentially associated with the WAVE complex subunit ABI1 when RAC1 is activated and downstream generation of branched actin is blocked. PPP2R1A is found to associate at the lamellipodial edge with an alternative form of WAVE complex, the WAVE Shell Complex, that contains NHSL1 instead of the Arp2/3 activating subunit WAVE, as in the canonical WAVE Regulatory Complex. PPP2R1A is required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement is abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impair WAVE Shell Complex binding and migration regulation, suggesting that the coupling of PPP2R1A to the WAVE Shell Complex is essential to its function. (10.1038/s41467-023-39276-w)
  DOI : 10.1038/s41467-023-39276-w
• N 2-methylguanosine modifications on human tRNAs and snRNA U6 are important for cell proliferation, protein translation and pre-mRNA splicing
  
  • Wang Can
  • Ulryck Nathalie
  • Herzel Lydia
  • Pythoud Nicolas
  • Kleiber Nicole
  • Guerineau Vincent
  • Jactel Vincent
  • Moritz Chloé
  • Bohnsack Markus T
  • Carapito Christine
  • Touboul David
  • Bohnsack Katherine E
  • Graille Marc
  Nucleic Acids Research, Oxford University Press, 2023, 51 (14), pp.7496-7519. Modified nucleotides in non-coding RNAs, such as 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 (Trm112 in Saccharomyces cerevisiae), but the interactome of this regulator and targets of its interacting MTases remain incompletely characterized. Here, we have investigated the interaction network of human TRMT112 in intact cells and identify three poorly characterized putative MTases (TRMT11, THUMPD3 and THUMPD2) as direct partners. We demonstrate that these three proteins are active N2-methylguanosine (m2G) MTases and that TRMT11 and THUMPD3 methylate positions 10 and 6 of tRNAs, respectively. For THUMPD2, we discovered that it directly associates with the U6 snRNA, a core component of the catalytic spliceosome, and is required for the formation of m2G, the last ‘orphan’ modification in U6 snRNA. Furthermore, our data reveal the combined importance of TRMT11 and THUMPD3 for optimal protein synthesis and cell proliferation as well as a role for THUMPD2 in fine-tuning pre-mRNA splicing. (10.1093/nar/gkad487)
  DOI : 10.1093/nar/gkad487