Biology subjects

Fedry, J.

Publications and source records attributed to Fedry, J..

6 recordsLinked to original sources

bioRxiv2026

In-cell structural insights into fungal ER stress responses

Approximately a third of a cells proteome is folded in the endoplasmic reticulum (ER). The accumulation of misfolded proteins in the ER results in ER stress. The unfolded protein response (UPR) has evolved to attenuate ER stress by increasing the cells folding capacity and reducing the folding load in the ER. In metazoans, the latter is notably achieved through mRNA translation inhibition. However, S. cerevisiae lacks the corresponding pathways, and its reliance on translational downregulation in response to ER stress remains largely unexplored. In this study we combined cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to investigate the effect of ER stress on the translational machinery of S. cerevisiae. At the ultrastructural level, we describe changes in organelle morphology, confirming ER volume expansion and the activation of autophagy. Further structural analysis reveals that ER stress triggers an increase in the relative abundance of inactive 80S ribosomes (up to 25% of the total), both in the cytosol and at the ER. Finally, we visualise the binding of the elongation factors eEF2, eIF5A and eEF3 to inactive ribosomes. Our work provides evidence that ER stress in S. cerevisiae leads to modest translation inhibition, which might contribute to alleviating ER stress.

molecular biology↗

bioRxiv2024

NAC-mediated ribosome localization regulates cell fate and metabolism in intestinal stem cells

Intestinal stem cells (ISCs) face the challenge of integrating metabolic demands with unique regenerative functions. Studies have shown an intricate interplay between metabolism and stem cell capacity, however it is still not understood how this process is regulated. Combining ribosome profiling and CRISPR screening in intestinal organoids, we show that RNA translation is at the root of this interplay. We identify the nascent polypeptide-associated complex (NAC) as a key mediator of this process, and show that it regulates ISC metabolism by relocalizing ribosomes to the mitochondria. Upon NAC inhibition, intestinal cells show decreased import of mitochondrial proteins, which are needed for oxidative phosphorylation, and, consequently, enable the cell to maintain a stem cell identity. Furthermore, we show that overexpression of NAC is sufficient to drive mitochondrial respiration and promote ISC identity. Ultimately, our results reveal the pivotal role of ribosome localization in regulating mitochondrial metabolism and ISC function. TeaserThe location of ribosomes in cells is regulated, and defines the fate of intestinal stem cells.

molecular biology↗

bioRxiv2023

Visualization of translation reorganization upon persistent collision stress in mammalian cells

Aberrantly slow mRNA translation leads to ribosome stalling and subsequent collision with the trailing neighbor. Ribosome collisions have recently been shown to act as stress sensors in the cell, with the ability to trigger stress responses balancing survival and apoptotic cell-fate decisions depending on the stress level. However, we lack a molecular understanding of the reorganization of translation processes over time in mammalian cells exposed to an unresolved collision stress. Here we visualize the effect of a persistent collision stress on translation using in situ cryo electron tomography. We observe that low dose anisomycin collision stress leads to the stabilization of Z-site bound tRNA on elongating 80S ribosomes, as well as to the accumulation of an off-pathway 80S complex possibly resulting from collision splitting events. We visualize collided disomes in situ, occurring on compressed polysomes and revealing a stabilized geometry involving the Z-tRNA and L1 stalk on the stalled ribosome, and eEF2 bound to its collided rotated-2 neighbor. In addition, non-functional post-splitting 60S complexes accumulate in the stressed cells, indicating a limiting Ribosome associated Quality Control clearing rate. Finally, we observe the apparition of tRNA-bound aberrant 40S complexes shifting with the stress timepoint, suggesting a succession of different initiation inhibition mechanisms over time. Altogether, our work visualizes the changes of translation complexes under persistent collision stress in mammalian cells, indicating how perturbations in initiation, elongation and quality control processes contribute to an overall reduced protein synthesis. SummaryUsing in situ cryo electron tomography we visualized the reorganization of mammalian translation processes during a persistent collision stress.

cell biology↗

bioRxiv2022

SEC62 and TMX4 control asymmetric autophagy of the nuclear envelope upon LINC complex disassembly

The endoplasmic reticulum (ER) is a dynamic organelle of nucleated cells that produces proteins, lipids and oligosaccharides. The volume and the activities of the ER are adapted to cellular needs. They are increased upon induction of unfolded protein responses (UPR 1) and are reduced upon activation of ER-phagy programs 2. A specialized domain of the ER, the nuclear envelope (NE), protects the cell genome with two juxtaposed lipid bilayers, the inner and outer nuclear membranes (INM and ONM). SUN proteins in the INM form disulfide-bonded Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes with NESPRIN proteins in the ONM. These complexes set and maintain the width of the periplasmic space (PS), a continuum of the ER lumen, below the 50 nm 3-5 and in yeast prevent transmission of ER volume variations to the PS 6,7. Here we report that expansion of the mammalian ER upon homeostatic perturbations is transmitted to the NE, where the ONM forms large bulges. The process is reverted on recovery of ER homeostasis, by asymmetric vesiculation and autophagic clearance of ONM portions. Remodeling of the mammalian NE requires TMX4-driven reduction of the intermolecular disulfide bond stabilizing LINC complexes, the LC3 lipidation machinery, and the autophagy receptor SEC62, identified here as the first mammalian nucleo-phagy receptor.

cell biology↗

bioRxiv2020

A human monoclonal antibody targeting a conserved pocket in the SARS-CoV-2 receptor-binding domain core

SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.

immunology↗

bioRxiv2018

Hybrid sequence-structure based HMM models leverage theidentification of homologous proteins: the example of class II fusion

We present a sequence-structure based method characterizing a set of functionally related proteins exhibiting low sequence identity and loose structural conservation. Given a (small) set of structures, our method consists of three main steps. First, pairwise structural alignments are combined with multi-scale geometric analysis to produce structural motifs i.e. regions structurally more conserved than the whole structures. Second, the sub-sequences of the motifs are used to build profile hidden Markov models (HMM) biased towards the structurally conserved regions. Third, these HMM are used to retrieve from UniProtKB proteins harboring signatures compatible with the function studied, in a bootstrap fashion.\n\nWe apply these hybrid HMM to investigate two questions related to class II fusion proteins, an especially challenging class since known structures exhibit low sequence identity (less than 15%) and loose structural similarity (of the order of 15[A] in lRMSD). In a first step, we compare the performances of our hybrid HMM against those of sequence based HMM. Using various learning sets, we show that both classes of HMM retrieve unique species. The number of unique species reported by both classes of methods are comparable, stressing the novelty brought by our hybrid models. In a second step, we use our models to identify 17 plausible HAP2-GSC1 candidate sequences in 10 different drosophila melanogaster species. These models are not identified by the PF[A]M family HAP2-GCS1 (PF10699), stressing the ability of our structural motifs to capture signals more subtle than whole Pfam domains.\n\nIn a more general setting, our method should be of interest for all cases functional families with low sequence identity and loose structural conservation.\n\nOur software tools are available from the FunChaT package of the Structural Bioinformatics Library (http://sbl.inria.fr).

bioinformatics↗