Biology subjects

Aupic, J.

Publications and source records attributed to Aupic, J..

7 recordsLinked to original sources

bioRxiv2025

The Covalent Binding of Splicing Inhibitors occurs via a Zinc- Assisted Enzymatic-like mechanism

Branch-site recognition is a pivotal event in spliceosome assembly, and branch-site antagonists disrupt this process, inducing a splicing rewiring that underlies much of their antitumor activity. Antagonists from the spliceostatin family covalently bind the SF3b complex nearby the branch-site pocket, forming an adduct with Cys26 of the PHF5A zinc finger while remarkably preserving Zn2+ coordination - an unconventional mode of covalent inhibition. Yet the molecular features that activate this normally protected ZnCys4 cysteine and initiate spliceostatins epoxide ring opening have remained obscure. Here, we combine extensive classical and QM/MM molecular dynamics simulations to map the full reactive trajectory of spliceostatin attachment to PHF5A. We identify two alternative noncovalent spliceostatin binding poses and show that only its thermodynamically favoured conformation pre-organizes the epoxide for nucleophilic attack. The local distortion of the ZnCys4 coordination sphere weakens the Zn-Cys26 bond, enabling water-Cys26 exchange and generating a highly reactive nucleophilic thiolate. Covalent bond formation is subsequently accelerated by an Asp34-Lys29 proton relay that activates the epoxide leaving group. Free-energy calculations confirm that the overall reaction is fast and strongly exergonic. Together, these findings provide a complete mechanistic framework for zinc-assisted inhibition of early spliceosomes by branch-site antagonists and advance our fundamental understanding of zinc finger reactivity.

biochemistry↗

bioRxiv2025

Structural flexibility of the human vault protein revealed by high-resolution cryo-EM and molecular dynamics simulations

Vaults are massive ribonucleoprotein complexes, highly conserved and abundant in eukaryotic cells, yet with unclear function. Their thin-walled barrel-shape architecture is composed of two symmetrical, antiparallel half-shells, each containing 39 copies of the major vault protein (MVP). The spacious lumen of the vault suggests a role in cellular transport. To facilitate cargo encapsulation and release, the vault is thought to open into two halves, yet the molecular mechanism governing vault opening remains elusive. Here, we combine cryogenic electron microscopy (cryo-EM) and multi-scale molecular dynamics (MD) simulations to reveal the structural factors giving flexibility to the human vault protein. Using cryo-EM, we identified two alternative conformational states of the human vault, along with the half-vault shell. MD simulations of these structures show extensive, breathing-like motions, porous solvent-exposed surfaces, and distinct structural variability between conformational states. The stable intermediates and the flexibility at the interface of the half vaults together suggest a possible mechanism for the dynamic assembly and disassembly of the vault.

biochemistry↗

bioRxiv2025

Recognition and catalytic mechanism of tRNA m3C methyltransferase METTL6

Post-transcriptional RNA modifications regulate the RNA structural and functional landscape, and thus play a central role in many cellular processes. Accordingly, aberrant RNA modification pathways contribute to a variety of human diseases, including cancer and neurological disorders. However, recognition and catalytic mechanisms of most RNA-modifying proteins remain obscure, due to sparse structural data. Recently, the cryogenic electron microscopy structure of N3-methyl-cytosine (m3C) methyltransferase METTL6 revealed seryl-tRNA synthetase (SerRS) as a cofactor for tRNASer selection, but left the cooperative role of SerRS and the reaction process undefined. Here, combining computer simulations and in vitro experiments, we define the mechanism of concerted substrate identification by METTL6 and SerRS, demonstrating that SerRS pre-organizes the tRNASer variable arm for METTL6 recruitment. We show that once METTL6 is stably docked onto the SerRS-tRNASer complex, thereby ensuring the precise alignment of the reactants, the methylation reaction proceeds spontaneously. Our results establish the catalytic mechanism of m3C methyltransferases and provide the foundation for rational design of targeted therapeutics.

biochemistry↗

bioRxiv2024

All-atom simulations elucidate the molecular mechanismunderlying RNA-membrane interactions

RNA-membrane interactions are starting to emerge as an important organizing force in both natural and synthetic biological systems. Notably, RNA molecules were recently discovered to be present on the extracellular surface of living cells, where they mediate intercellular signalling. Furthermore, RNA-membrane interactions influence the efficacy of lipid-based RNA delivery systems. However, the molecular terms driving RNA localisation at the membrane remain poorly understood. In this work, we investigate how RNA-phospholipid membrane interactions occur, by means of all-atom simulations. We find that among the four RNA nucleobases guanine exhibits the strongest interaction with the membrane due to extensive hydrogen bond formation. Additionally, we show that intra-RNA base pairing present in organised RNA structures significantly hinders RNA binding to the membrane. Elucidating the molecular details of RNA-membrane association will importantly contribute to improving the design of RNA-based drugs as well as lipid-based RNA delivery systems and to parsing out RNA transport and localisation mechanisms.

biophysics↗

bioRxiv2021

The Conformational Plasticity of the Selectivity Filter Methionines Controls the In-Cell Cu(I) Uptake through the CTR1 transporter

Copper is a trace element vital to many cellular functions. Yet its abnormal levels are toxic to cells, provoking a variety of severe diseases. The high affinity Copper Transporter 1 (CTR1), being the main in-cell copper (Cu(I)) entry route, tightly regulates its cellular uptake via a still elusive mechanism. Here, all-atoms simulations unlock the molecular terms of Cu(I) transport in eukaryotes disclosing that the two Methionine triads, forming the selectivity filter, play an unprecedented dual role both enabling selective Cu(I) transport and regulating its uptake-rate thanks to an intimate coupling between the conformational plasticity of their bulky side chains and the number of bound Cu(I) ions. Namely, the Met residues act as a gate reducing the Cu(I) import-rate when two ions simultaneously bind to CTR1. This may represent an elegant autoregulatory mechanism through which CTR1 protects the cells from excessively high, and hence toxic, in-cell Cu(I) levels. Overall, these outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/467269v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@75fbe6org.highwire.dtl.DTLVardef@1ece24eorg.highwire.dtl.DTLVardef@fda950org.highwire.dtl.DTLVardef@1770038_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism and screen inhibition of viral spike protein-mediated fusion under the biosafety level 1conditions.

synthetic biology↗

bioRxiv2020

Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein

Effective and safe vaccines against SARS-CoV-2 are highly desirable to prevent casualties and societal cost caused by Covid-19 pandemic. The receptor binding domain (RBD) of the surface-exposed spike protein of SARS-CoV-2 represents a suitable target for the induction of neutralizing antibodies upon vaccination. Small protein antigens typically induce weak immune response while particles measuring tens of nanometers are efficiently presented to B cell follicles and subsequently to follicular germinal center B cells in draining lymph nodes, where B cell proliferation and affinity maturation occurs. Here we prepared and analyzed the response to several DNA vaccines based on genetic fusions of RBD to four different scaffolding domains, namely to the foldon peptide, ferritin, lumazine synthase and {beta}-annulus peptide, presenting from 6 to 60 copies of the RBD on each particle. Scaffolding strongly augmented the immune response with production of neutralizing antibodies and T cell response including cytotoxic lymphocytes in mice upon immunization with DNA plasmids. The most potent response was observed for the 24-residue {beta}-annulus peptide scaffold that forms large soluble assemblies, that has the advantage of low immunogenicity in comparison to larger scaffolds. Our results support the advancement of this vaccine platform towards clinical trials.

immunology↗