Biology subjects

Favier, A.

Publications and source records attributed to Favier, A..

11 recordsLinked to original sources

bioRxiv2026

Conserved upper thermal limits and small safety margins in soil copiotrophic bacteria.

One of the key uncertainties in climate change models is how microbes will adapt to rising temperatures. Large-scale comparisons of bacterial thermal performances show a clear boundary between mesophiles and thermophiles. Here, we investigated whether phylogenetic constraints limit the adaptive potential of bacteria to warming soils. Focusing on copiotrophs within Gammaproteobacteria, we found that both thermal optima and upper thermal limits are constrained; variation in these traits decreases above 42{degrees}C across the phylogeny, with minimal influence of present-day bioclimatic variables. This, along with the reduced thermal safety margins found in fluctuating hot climates, suggests that many isolates may already be maladapted to local temperature variability. Our findings indicate that these constraints interact with the geometry of thermal performance curves, imposing a trade-off between high-temperature performance and the risk of substantial fitness losses above Topt. Overall, this work underscores potential limits to thermal adaptation and their implications for bacterial fitness.

evolutionary biology↗

bioRxiv2025

A Fast and efficient strategy for the NMR assignment of Fab methyl groups

Owing to their high specificity and therapeutic effectiveness, monoclonal antibodies (mAbs) have rapidly become one of the leading classes of biologic drugs used to treat critical illnesses. The antigen-binding fragment (Fab) of mAbs plays a key role in the antigen recognition, so its structural characterization is essential, as even a slight change to its Higher Order Structure (HOS) can impact the antibodys potency. Recently, 2D methyl NMR has been introduced as a powerful method to assess both the structure and integrity of therapeutic Fab fragments. However, the identification of methyl group resonances in NMR spectra remains rare since Fabs are large heterodimers of [~]50 kDa. Here, we present the methyl group assignment of an IgG1 Fab produced in a cell-free system with an optimal isotope labelling. We first assigned 99% of the alanine, isoleucine, leucine, methionine, and valine methyl groups of the therapeutic Fab targeting LAMP1 antigen. Building on this assignment, we propose a "divide and conquer" strategy that exploits sequence identities to rapidly assign methyl groups of other IgG1 Fabs. We demonstrate that the assignment of the Fabs constant region can easily be transferred from one IgG1 to another and that the variable part of a new Fab can be assigned using smaller uniformly 15N,13C-labelled constructs. We applied our strategy to ipilimumabs Fab and, using the assignment of ipilimumabs variable part and Fab anti-LAMP1s constant part, we could transfer the assignment of 89% of the methyl-containing amino acids to the entire ipilimumab Fab without having to produce deuterated samples. This assignment strategy can be generalised to any other IgG1 Fabs provided that their constant regions are identical and the strategy can be adapted to accommodate the expression levels of the different variable domains. This new method drastically facilitates the Fab assignment process, making it suitable for the pharmaceutical timeline.

biochemistry↗

bioRxiv2025

Identification of uranyl-binding proteins in Arabidopsis thaliana cells exposed to uranium: Insights from a metalloproteomic analysis and characterization of Glycine-Rich RNA-binding Protein 7 (GRP7)

Uranium (U) is a naturally occurring radionuclide, chemotoxic for living organisms. To identify proteins that could be cellular targets for U in plants, we used metalloproteomic approaches combining column chromatographic fractionation analyses, protein identification by high-resolution mass spectrometry shotgun proteomics, and metal quantification by induced coupled plasma mass spectrometry. We identified 57 candidate proteins for uranyl (U(VI)) binding in cultured Arabidopsis thaliana cells. One of these proteins, the Glycine-Rich RNA-binding Protein 7 (GRP7) is a RNA-binding protein involved in various developmental processes and responses to biotic and abiotic stress. Recombinant GRP7 was purified from overproducing bacteria and subjected to further biochemical characterization. First, we showed that GRP7 binds U(VI) with a 1:2 (protein:metal) stoichiometry in vitro. Next, we analyzed its structural properties by solution-state nuclear magnetic resonance spectroscopy. This allowed us to gain insight into the molecular dynamics of the protein-metal interaction and to identify residues involved in U(VI)-binding in the two sites. Finally, we observed that U(VI) binding interferes with nucleic acid binding to the protein RNA-binding domain, suggesting that U(VI) binding to GRP7 contributes to U toxicity in plants. HIGHLIGHTSO_LI57 candidate uranyl-binding proteins were identified from Arabidopsis cells exposed to uranyl, of which the RNA-binding protein GRP7. C_LIO_LIIn vitro, recombinant GRP7 binds 2 uranyl ions within the RNA recognition motif domain. C_LIO_LIAmino acid residues involved in uranyl binding in both binding sites were identified. C_LIO_LICompetition between specific oligonucleotide and uranyl for binding suggests the implication of GRP7 in uranium toxicity, as a cellular target. C_LI

biochemistry↗

bioRxiv2024

Light-dependent flavin redox and adduct states control the conformation and DNA binding activity of the transcription factor EL222

The activity of the transcription factor EL222 is regulated through protein-chromophore adduct formation, interdomain dynamics, oligomerization and protein-DNA interactions, all triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV/visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using non-canonical amino acids. The observation of only subtle atomic displacements between crystal structures of EL222 with and without blue-light back-illumination, was confirmed by NMR data indicating no major changes in secondary structure and fold compactness. Kinetic experiments in solution provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently-bound N5 protonated, and non-covalently-bound hydroquinone forms of FMN, respectively. Molecular modeling revealed differential dynamics and domain separation times arising from the three FMN states (oxidized, adduct, and reduced). Furthermore, while the dark state is largely monomeric, both lit states undergo slow monomer-dimer exchange. The photoinduced loss of -helicity, seen by infrared difference spectroscopy, was ascribed to dimeric EL222 species. Unexpectedly, NMR revealed that all three EL222 species (dark, lit1, lit2) can associate with DNA to some extent, but only under illumination a high population of stable complexes is obtained. Overall, we propose a refined model of EL222 photo-activation where photoinduced changes in the oxidation state of FMN and thioadduct formation shift the population equilibrium towards an open conformation that favors self-association and DNA-binding. Significance StatementFlavin-binding light-oxygen-voltage (LOV) proteins constitute a prominent example of highly evolved chromophore-containing proteins that convert light into biochemical changes in the cell. However, it is not well understood how blue-light orchestrates changes in LOV structure and function. Here we show that the dynamics, oligomerization and DNA-binding properties of the photocontrolled transcription factor EL222 are dependent on both the flavin redox state and thioadduct formation. In the dark, monomeric EL222 forms transient encounter complexes with DNA. Under illumination, two distinct lit states are sequentially generated, termed lit1 and lit2, that are both able to assemble into EL222:DNA (2:1) complexes. Our results reveal the coupling between flavin photochemistry (protonation and covalent linkage) and fold stability in EL222 and potentially other flavoproteins.

biochemistry↗

bioRxiv2023

Predictive microbial community changes across a temperature gradient

A central challenge in community ecology is predicting the effects of abiotic factors on community assembly. In particular, microbial communities play a central role in the ecosystem, but we do not understand how changing factors like temperature are going to affect community composition or function. One of the challenges is that we do not understand the mechanistic impacts of temperature on different metabolic strategies, nor how this metabolic plasticity could impact microbial interactions. Dissecting the contribution of environmental factors on microbial interactions in natural ecosystems is hindered by our understanding of microbial physiology and our ability to disentangle interactions from sequencing data. Studying the self-assembly of multiple communities in synthetic environments, here we are able to predict changes in microbial community composition based on metabolic responses of each functional group along a temperature gradient. This research highlights the importance of metabolic plasticity and metabolic trade-offs in predicting species interactions and community dynamics across abiotic gradients.

ecology↗

bioRxiv2022

Structural analysis shows that the BIR2 domain of E3 ligase XIAP binds across the RIP2 kinase dimer interface

NOD1 and NOD2 are innate immune system pattern recognition receptors that play a key role in the protection from bacterial infections and in the maintenance of gastro-intestinal homeostasis. Dysregulation of NOD signalling pathways promotes chronic inflammatory diseases such as inflammatory bowel disease. RIP2, which contains a kinase domain (RIP2K) and a CARD domain, is the immediate downstream signalling partner in the NOD pathway and inhibition of its kinase activity and ubiquitination are promising strategies to address these inflammatory diseases. However, recent work indicates that the phosphorylation activity of RIP2K is dispensable for signalling and that inhibitors of both RIP2K activity and RIP2 ubiquitination prevent the essential interaction between RIP2K and the BIR2 domain of XIAP, the key RIP2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined Native mass spectrometry, NMR, cryo-electron microscopy and Alphafold2 predictions to determine the structure of the RIP2K-XIAP BIR2 complex with 2:1 stoichiometry. The structure shows that complex formation requires that the kinase is in an active-like (i.e. C-helix IN) and dimeric conformation and explains both inhibitory mechanisms. It also shows why phosphorylation of the kinase activation loop is dispensable for signalling and reveals the structural role of RIP2K-K209 residue in the RIP2K-XIAP BIR2 interaction. Importantly, our results clarify the features of the RIP2K conformation essential for its role as a scaffold protein for ubiquitination.

biochemistry↗

bioRxiv2022

Visualizing the Transiently Populated Closed-State of Human HSP90 ATP Binding Domain

HSP90 are abundant molecular chaperones, assisting the folding of several hundred client proteins, including substrates involved in tumor growth or neurodegenerative diseases. A complex set of large ATP-driven structural changes occurs during HSP90 functional cycle. However, the existence of such structural rearrangements in apo HSP90 has remained unclear. Here, we identified a metastable excited state in the isolated HSP90 ATP binding domain. We used solution NMR and mutagenesis to characterize structures of both ground and excited states. We demonstrated that in solution the HSP90 ATP binding domain transiently samples a functionally relevant ATP-lid closed state, distant by more than 30 [A] from the ground state. NMR relaxation and molecular dynamics were combined to characterize the energy landscape corresponding to the transition between these interconverting states. The precise description of the dynamics and structures sampled by human HSP90 ATP binding domain is a paramount piece of information for the future design of new therapeutic ligands.

biophysics↗

bioRxiv2020

Modeling disease-correlated TUBA1A mutation in budding yeast reveals a molecular basis for tubulin dysfunction

Malformations of cortical development (MCD) of the human brain are a likely consequence of defective neuronal migration, and/or proliferation of neuronal progenitor cells, both of which are dictated in part by microtubule-dependent transport of various cargoes, including the mitotic spindle. Throughout the evolutionary spectrum, proper spindle positioning depends on cortically anchored dynein motors that exert forces on astral microtubules emanating from spindle poles. A single heterozygous amino acid change, G436R, in the conserved TUBA1A -tubulin gene was reported to account for MCD in patients. The mechanism by which this mutation disrupts microtubule function in the developing cerebral cortex is not understood. Studying the consequence of tubulin mutations in mammalian cells is challenging partly because of the large number of -tubulin isotypes expressed. To overcome this challenge, we have generated a budding yeast strain expressing the mutated tubulin (Tub1G437R in yeast) as one of the main sources of -tubulin (in addition to Tub3, another -tubulin isotype in this organism). Although viability of the yeast was unimpaired by this mutation, they became reliant on Tub3, as was apparent by the synthetic lethality of this mutant in combination with tub3{Delta}. We find that Tub1G437R assembles into microtubules that support normal G1 activity, but lead to enhanced dynein-dependent nuclear migration phenotypes during G2/M, and a consequential disruption of spindle positioning. We find that this mutation impairs the interaction between She1 - a negative regulator of dynein - and microtubules, as was apparent from a yeast two-hybrid assay, a co-sedimentation assay, and from live cell imaging. We conclude that a weaker interaction between She1 and Tub1G437R-containing microtubules results in enhanced dynein activity, ultimately leading to the spindle positioning defect. Our results provide the first evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation, and sheds light on a mechanism that may be causative of neurodevelopmental diseases.

cell biology↗

bioRxiv2020

Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis

The initial greening of angiosperm occurs upon light-activation of photoreceptors that trigger photomorphogenesis followed with the development of chloroplasts. In these semi-autonomous organelles, the construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here we show that PAP8, as an essential subunit of the plastid-encoded RNA polymerase, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 is localized and active in both plastids and the nucleus and particularly essential for the formation of late photobodies. In the albino pap8 mutant, phytochrome-mediated signalling is altered, PIFs are maintained, HY5 is not stabilized, and GLK1 expression is impaired. PAP8 translocates into plastids losing its pre-sequence, interacts with the PEP, and using an unknown route or a retrograde transport, reaches the nucleus where it has the ability to interact with pTAC12/HMR/PAP5. Since PAP8 is required for the phytochrome-B-mediated signalling cascade and the reshaping of the PEP, it may coordinate nuclear gene expression with the PEP-driven chloroplastic gene expression during chloroplast biogenesis.

plant biology↗

bioRxiv2019

Common homozygosity for predicted loss-of-function variants reveals both redundant and advantageous effects of dispensable human genes

Humans homozygous or hemizygous for variants predicted to cause a loss of function of the corresponding protein do not necessarily present with overt clinical phenotypes. However, the set of effectively dispensable genes in the human genome has not yet been fully characterized. We report here 190 autosomal genes with 207 predicted loss-of-function variants, for which the frequency of homozygous individuals exceeds 1% in at least one human population from five major ancestry groups. No such genes were identified on the X and Y chromosomes. Manual curation revealed that 28 variants (15%) had been misannotated as loss-of-function, mainly due to linkage disequilibrium with different compensatory variants. Of the 179 remaining variants in 166 genes (0.82% of 20,232 genes), only 11 alleles in 11 genes had previously been confirmed experimentally to be loss-of-function. The set of 166 dispensable genes was enriched in olfactory receptor genes (41 genes), but depleted of genes expressed in a wide range of organs and in leukocytes. The 125 dispensable non-olfactory receptor genes displayed a relaxation of selective constraints both between species and within humans, consistent with greater redundancy. In total, 62 of these 125 genes were found to be dispensable in at least three human populations, suggesting possible evolution toward pseudogenes. Out of the 179 common loss-of-function variants, 72 could be tested for two neutrality selection statistics, and eight displayed robust signals of positive selection. These variants included the known FUT2 mutant allele conferring resistance to intestinal viruses and an APOL3 variant involved in resistance to parasitic infections. Finally, the 41 dispensable olfactory receptor genes also displayed a strong relaxation of selective constraints similar to that observed for the 341 non-dispensable olfactory receptor genes. Overall, the identification of 166 genes for which a sizeable proportion of humans are homozygous for predicted loss-of-function alleles reveals both redundancies and advantages of such deficiencies for human survival.\n\nSignificance statementHuman genes homozygous for seemingly loss of function (LoF) variants are increasingly reported in a sizeable proportion of individuals without overt clinical phenotypes. Here, we found 166 genes with 179 predicted LoF variants for which the frequency of homozygous individuals exceeds 1% in at least one of the populations present in databases ExAC and gnomAD. This set of putatively dispensable genes showed relaxation of selective constraints suggesting that a large number of these genes are undergoing pseudogenization. Eight of the common LoF variants displayed robust signals of positive selection including two variants located in genes involved in resistance to infectious diseases. The identification of dispensable genes will allow identifying functions that are, at least nowadays, redundant, or possibly advantageous, for human survival.

genetics↗

bioRxiv2018

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

Atomic-resolution structure determination is the key requirement for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enabled determination of the high-resolution structure of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 [A] by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and specifically labelled methyl groups, and a 4.1 [A] resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution (up to 8 [A]) cryo-EM data are available, thus paving avenues for the structure determination of challenging biological assemblies.

biophysics↗