Biology subjects

Brugiere, S.

Publications and source records attributed to Brugiere, S..

11 recordsLinked to original sources

bioRxiv2026

Investigating Histone Lysine Acetylation and Acetyl-methylation in Mouse Brain using Enhanced Trimethylacetic Anhydride Derivatization Protocol

Histone post-translational modifications (PTMs) are key regulators of gene expression, and mass spectrometry (MS) provides a powerful platform for their characterization and quantification. However, conventional histone sample preparation using derivatization of non-modified lysines by propionic anhydride (PA) presents analytical limitations, including non-detection of short hydrophilic peptides, incomplete chromatographic separation of positional isomers, and masking of endogenous lysine propionylation and butyrylation. Here, we optimized a trimethylacetic anhydride (TMA)-based derivatization workflow to improve histone PTM analysis. Compared with PA derivatization, TMA enhanced the detection of acetylated peptides, particularly short and hydrophilic species, although peptide-dependent ionization biases affected relative peptidoform quantification. We further evaluated the suitability of TMA derivatization for investigating endogenous lysine propionylation and butyrylation. Unexpectedly, we identified lysine acetyl-methylation (Kacme) at seven sites on histone H3 and validated these assignments by targeted MS using synthetic reference peptides. To investigate the metabolic dynamics of Kacme, we performed in vivo isotope tracing in mice administered 13C-labeled glucose. Unlike canonical lysine acetylation, Kacme exhibited minimal incorporation of glucose-derived acetyl groups over 2h, suggesting substantially slower turnover of its acetyl moiety. Collectively, these findings highlight the analytical advantages and quantitative limitations of TMA-based histone derivatization, while revealing Kacme as a metabolically distinct histone modification and expanding the scope of MS-based histone PTM analysis.

biochemistry↗

bioRxiv2025

Both L-lactyl and D-lactyl enantiomers modify histones in mouse testis

Dynamic histone post-translational modifications are crucial to precisely orchestrate gene expression programs. The recently discovered histone lysine lactylation has already been explored in various pathological contexts, but less in normal tissues. This modification exists as two enantiomers, L- and D-lactylation; the first one may more likely modify histones due to abundant L-lactate produced by glycolysis. Here we report the identification by proteomics of L- and D-lactylation on lysines of histones H3 and H4 in mouse testis. We developed a targeted proteomic analysis of histone peptides using synthetic sequences modified by L- or D-lactyl, to acquire reliable identification and quantification data. Some histone peptides bearing either enantiomer are separated by reversed-phase chromatography. Interestingly, while several lysines of H3 and H4 exhibit a balanced amount of both enantiomers, residues 18 and 79 from histone H3 (H3K18 and H3K79) appear to be significantly more D-lactylated, while H3K23 is more L-lactylated. The stoichiometry of lactylation is low over the whole sequence of H3 and H4, representing 0.01 to 0.5%, which contrasts with acetylation whose abundance substantially varies between lysines. Interestingly, lactylation is more abundant than acetylation on the C-terminal half of H3 and H4. These results suggest a mechanism producing a mixture of the two enantiomers of lactate, or of a more direct substrate for lactylation, and supporting a site-selective addition of the enantiomers.

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

Mind Your Spectra: Points to be Aware of when Validating the Identification of Isobaric Histone Peptidoforms

Mass spectrometry (MS) has become a central technique to identify and quantify post-translational modifications (PTMs), overcoming limitations of antibody-based methods. Histones get dynamically modified by diverse chemical groups, particularly on their numerous lysine residues, to fine-tune all DNA-templated processes. Reliable identification of histone PTMs remains challenging and still requires manual data curation. This study focused on the Lys27-Arg40 stretch of histone H3, considered four sequence variants, an increasing number of lysine PTMs and artifacts coming from histone sample processing, which resulted in many peptides with the same atomic composition. Our analysis revealed the value of low-mass b1 and cyclic immonium fragment ions to validate identification of the distinct peptidoforms. We examined how MS/MS spectra are transformed by common software tools during the conversion of RAW files into peak lists, and highlighted how some parameters may erase the informative low-mass fragments. We established the fragmentation profiles and retention times for forty H3 K27-R40 variantxPTM combinations, including the mouse-specific variants H3mm7 and H3mm13, and targeted their detection in histone samples extracted from mouse testis and brain via a scheduled parallel reaction monitoring (PRM) analysis. The transcripts of these two mousespecific variants were reported to be highly abundant in these tissues and the corresponding proteins may seem to be identified by data-dependent analyses. However, we only detected very low levels of the unmodified form of H3mm7 and found no trace of H3mm13 by PRM. Our work contributes to reliably deciphering the histone code shaped by distinct sequence variants and numerous combinations of PTMs.

biochemistry↗

bioRxiv2024

The plastid-encoded RNA polymerase structures a logistic chain for light-induced photosynthesis

The chloroplast is the semi-autonomous organelle of eukaryotes that performs photosynthesis. In higher plants, chloroplast biogenesis depends on a tight transcriptional coordination of both nuclear- and-plastid photosynthesis-associated genes. The plastid-encoded RNA-polymerase (PEP) is composed of a plastid-encoded catalytic core, similar to multi-subunit RNA polymerases, bound to fifteen nuclear-encoded PEP-associated proteins (PAPs). The binding of all the PAPs to the catalytic core is essential for plastid transcription of photosynthesis-associated genes. Our cryo-electron microscopy structure of the native 21-subunit PEP from Sinapis alba reveals the distinctive patterning of PAP interactions, which evolved upon the ancestral cyanobacterial catalytic core acting as a scaffold. Using PAP8 in planta as bait for affinity purification and proximity labeling, we provide the protein landscapes surrounding the PEP and other PAP8-interacting complexes at the transition from skotomorphogenesis to photomorphogenesis. The data highlight multiple functional couplings in which plastid transcription is at the beginning of a spatial logistic chain, extending from transcription to the assembly of the photosynthetic apparatus into the thylakoids. In addition, dark-specific interactions between photoreceptors and PAP8 establish a physical link between an integrated light signaling and plastid functions.

plant biology↗

bioRxiv2024

AtVPS13M1 is involved in lipid remodeling in low phosphate and is located at the mitochondria surface in plants

VPS13 are conserved lipid transporters with multiple subcellular localizations playing key roles in many fundamental cellular processes. While the localization and function of VPS13 have been extensively investigated in yeast and animals, little is known about their counterparts in plants, particularly regarding their role in stress response. In this study, we characterized AtVPS13M1, one of the four VPS13 paralogs of the flowering plant Arabidopsis thaliana. We show that AtVPS13M1 binds and transports glycerolipids with a low specificity in vitro. AtVPS13M1 interferes with phospholipids degradation in response to phosphate starvation, a nutrient stress that triggers a massive remodeling of membrane lipids. AtVPS13M1 is mainly expressed in young dividing and vascular tissues. Finally, we show that AtVPS13M1 is mainly located at the surface of mitochondria in leaves. Overall, our work highlights the conserved role in lipid transport of VPS13 in plants, reveals their importance in nutrient stress response and opens important perspectives for the understanding of lipid remodeling mechanisms and for the characterization of this protein family in plants.

cell biology↗

bioRxiv2024

Photoreceptor-induced LHL4 protects photosystem II in Chlamydomonas reinhardtii

Photosynthesis, the fundamental process using light energy to convert CO2 to organic matter, is vital for life on Earth. It relies on capturing light through light-harvesting complexes in evolutionarily well-conserved photosystems (PS) I and II and on the conversion of light energy into chemical energy. Composition and organization of both photosystem core complexes are well conserved across evolution. PSII is particularly sensitive to photodamage but benefits from a large diversity of photoprotective mechanisms, finely tuned for the specific light conditions. Light Harvesting Complex protein family members (LHC and LHC-like families) have acquired a dual function during evolution. Members of the LHC antenna complexes of photosystems capture light energy whereas others dissipate excess energy that cannot be harnessed for photosynthesis. This process mainly occurs through non photochemical quenching (NPQ). In this work, we focus on the LHL4 protein, which is a LHC-like protein induced by UV-B and blue light photoreceptor signaling pathways in the model green microalgae Chlamydomonas reinhardtii. We demonstrate that alongside established NPQ effectors, LHL4 plays a key role in photoprotection, preventing singlet oxygen accumulation in PSII and promoting cell survival upon light stress. LHL4 protective function is distinct from that of NPQ-related proteins, as it specifically and uniquely binds to the transient monomeric form of the core PSII complex, safeguarding its integrity. LHL4 characterization expands our understanding of the interplay between light harvesting and photoprotection mechanisms upon light stress in photosynthetic microalgae.

plant biology↗

bioRxiv2024

Phototropin connects blue light perception to starch metabolism in green algae

In photosynthetic organisms light acts as an environmental signal to control their development and physiology, and as energy source to drive the conversion of CO2 into carbohydrates used for growth or storage. The main storage carbohydrate in green algae is starch, which accumulates during the day and is broken down at night to meet cellular energy demands. The signalling role of light quality in the regulation of starch accumulation remains unexplored. Here, we identify PHOTOTROPIN-MEDIATED SIGNALLING KINASE 1 (PMSK1) as a key regulator of starch metabolism in Chlamydomonas reinhardtii. In its phosphorylated form (PMSK1-P), it activates GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAP1), promoting starch biosynthesis. We show that blue light, perceived by PHOTOTROPIN, induces PMSK1 dephosphorylation that in turn represses GAP1 mRNA levels and reduces starch accumulation. These findings reveal a novel blue light-mediated signaling pathway that advances our understanding of photoreceptor-controlled carbon metabolism in microalgae. One-Sentence SummaryBlue light perception by PHOTOTROPIN triggers kinase-mediated signaling to inhibit starch accumulation in the green alga Chlamydomonas.

plant biology↗

bioRxiv2023

The α/β hydrolase domain-containing protein 1 (ABHD1) acts as a lysolipid lipase and is involved in lipid droplet formation

Lipid droplets (LDs) are the major sites of lipid and energy homeostasis. However, few LD biogenesis proteins have been identified. Here, using Chlamydomonas as a model, we show that ABHD1, a member of the /{beta} hydrolase domain-containing protein family, is a novel type of LD-associated protein which stimulates LD formation through two distinct actions on the LD surface, one enzymatic and the other structural. ABHD1 was localized to LD surface in Chlamydomonas cells. The knockout mutants contained similar amounts of triacylglycerols (TAG) but their LDs showed an increased content in lyso- derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). Over-expression of ABHD1 in Chlamydomonas induced LD formation and boosted TAG content, suggesting a key role in LD biogenesis. The purified recombinant ABHD1 protein hydrolyzed lyso-DGTS, producing a free fatty acid and a glyceryltrimethylhomoserine moiety. In vitro experiments using droplet- embedded vesicles showed that ABHD1 promoted LD emergence. Taken together, these results identify ABHD1 as a new player in LD formation by its lipase activity on lyso-DGTS and by its distinct biophysical property. This study further suggests that lipases targeted to LDs and able to act on their polar lipid coat may be interesting tools to promote LD assembly in eukaryotic cells. Significant statementLipid droplets are subcellular organelles specialized for triacylglycerol storage. Their dynamic turnover is key to managing energy homeostasis in response to cell cycle states and environmental cues. To gain insights into LD biogenesis, we characterized a putative /{beta}- hydrolase (ABHD1) in the model algae Chlamydomonas reinhardtii and show it is located at the LD surface. We found that ABHD1 overexpression promotes LD formation and acts as a lipase mainly on lyso derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), the major lipid constituent of the LD hemi-membrane. We also show that ABHD1 has a remarkable biophysical property favoring LD budding. This work thus identifies a novel type of lipase acting on betaine lipid and provides a first example of a protein with a dual function nvolved in LD formation.

biochemistry↗

bioRxiv2022

Proteomics unveil a central role for peroxisomes in butyrate assimilation of the heterotrophic Chlorophyte alga Polytomella sp.

Volatile fatty acids found in effluents of the dark fermentation of biowastes can be used for mixotrophic growth of microalgae, improving productivity and reducing the cost of the feedstock. Microalgae can use the acetate in the effluents very well, but butyrate is poorly assimilated and can inhibit growth above 1 gC.L-1. The non-photosynthetic chlorophyte alga Polytomella sp. SAG 198.80 was found to be able to assimilate butyrate fast. To decipher the metabolic pathways implicated in butyrate assimilation, quantitative proteomics study was developed comparing Polytomella sp. cells grown on acetate and butyrate at 1 gC.L-1. After statistical analysis, a total of 1772 proteins were retained, of which 119 proteins were found to be overaccumulated on butyrate vs. only 46 on acetate, indicating that butyrate assimilation necessitates additional metabolic steps. The data show that butyrate assimilation occurs in the peroxisome via the {beta}-oxidation pathway to produce acetyl-CoA and further tri/dicarboxylic acids in the glyoxylate cycle. Concomitantly, reactive oxygen species defense enzymes as well as the branched amino acid degradation pathway were strongly induced.Although no clear dedicated butyrate transport mechanism could be inferred, several membrane transporters induced on butyrate are identified as potential condidates. Metabolic responses correspond globally to the increased needs for central cofactors NAD, ATP and CoA, especially in the peroxisome and the cytosol.

microbiology↗

bioRxiv2022

Boosting chloroplast ribosome biogenesis by a plastidial DEAD-box RNA helicase is critical for high light acclimation

Photosynthetic organisms have developed sophisticated strategies to fine-tune light energy conversion to meet the metabolic demand, thereby optimizing growth in fluctuating light environments. Although mechanisms such as energy dissipation, photosynthetic control, or the photosystem II (PSII) damage and repair have been widely studied, little is known about the regulation of protein synthesis capacity during light acclimation. By screening a Chlamydomonas reinhardtii insertional mutant library using chlorophyll fluorescence imaging, we isolated a high chlorophyll fluorescence mutant (hf0) defected in a gene encoding a putative plastid targeted DEAD-box RNA helicase called CreRH22. CreRH22 is rapidly induced upon illumination and belongs to the GreenCut, a set of proteins specific to photosynthetic organisms. While photosynthesis is slightly affected in the mutant under low light (LL), exposure to high light (HL) induces a marked decrease in both PSII and PSI, and a strong alteration of the light-induced gene expression pattern. These effects are explained by the inability of hf0 to increase plastid ribosome amounts under HL. We conclude that CreRH22, by promoting ribosomal RNA precursor maturation in a light-dependent manner, enables the assembly of extra-ribosomes required to synthesize photosystem subunits at a higher rate, a critical step in the acclimation of algae to HL.

plant biology↗