Biology subjects

Bourgeois, B.

Publications and source records attributed to Bourgeois, B..

7 recordsLinked to original sources

bioRxiv2026

A histidine switch controls the pH-responsive self-assembly of a helical protein filament

Self-assembling helical protein filaments underlie diverse biological processes, from signaling pathways to cell motility. Encoding tunable self-assembly into the sequences of filamentous proteins remains a major challenge. Here, we discovered that the caspase-9 CARD can natively self-assemble into helical filaments in a pH-regulated manner. We defined the determinants of filament assembly using an integrative structural, biophysical, and computational approach. Using NMR spectroscopy, we found that the protonation of a single histidine residue near an N-terminal helix dipole, H38, regulates the pH-dependent self-assembly process. Charge-altering mutations at this site tune thermodynamic stability and filament self-assembly across solution and pH conditions. We solved 3.3- and 3.5-[A] cryo-EM structures of the wild-type and H38R filaments, respectively, which show H38 positioned directly at a filament interface. Molecular dynamics simulations show that H38 functions as a molecular switch, whereby protonation rotates its positively charged side-chain toward solvent and away from the partial-positive charge at the N-terminal helix dipole. This reflects a fine balance between stabilizing intermolecular association and a destabilizing intramolecular electrostatic clash at the helix dipole. More broadly, across 350 helix-containing protein domains we identified electrostatic contributions to protein stability near helix dipoles by integrating AlphaFold2 predictions with deep mutational scanning data. Together, our results identify a native, pH-sensitive histidine switch that regulates a self-assembling helical protein filament. Our results establish a mechanism by which charge-altering mutations near helical N-termini can be engineered to control side-chain rotamers, protein stability, and self-assembly.

biophysics↗

bioRxiv2026

TDP-43 self-assembly is regulated by its disordered NLS-region

Cytosolic inclusions of the RNA-binding protein TDP-43 are a pathological hallmark of several neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia. Cellular or animal model systems often use TDP-43 mutated in its nuclear localization signal (NLS) to study its cytosolic mislocalization and aggregation. Here we show that the disordered NLS-region, in particular the basic amino acids, are crucial for self-assembly of full-length TDP-43 across size scales, ranging from small clusters to visible condensates and aggregates. Molecular dynamics simulations and NMR studies suggest that the NLS-region engages in inter-chain interactions with C-terminal aromatic residues as well as RRM1 and NTD interactions. We further demonstrate that a minimal NLS mutation (K82A) preserves TDP-43 condensation in vitro and in cells, while commonly used NLS mutations yield partially or strongly reduced self-assembly behaviors. Our data highlight TDP-43 K82A as ideal model system to study cytosolic TDP-43 aggregation in cell and animal models.

biochemistry↗

bioRxiv2026

The sequence context of RG/RGG motifs determines condensate formation, transportin-1 binding and chaperoning

Intrinsically disordered arginine-glycine-rich (RG/RGG) regions are highly abundant in the eukaryotic proteome. Proteins containing these motifs participate in fundamental cellular processes, including nuclear import, transcriptional regulation, biomolecular condensate formation, and apoptosis. Mutations or dysfunction of RG/RGG proteins have been implicated in neurodegenerative diseases and cancer. Although some RG/RGG proteins have been shown to drive condensate formation, localize to membrane-less organelles, interact with nuclear import receptors, or undergo arginine methylation, these properties are not shared uniformly across the proteome. The considerable diversity in RG/RGG motif length and amino acid composition raises the question of which sequence features determine their functional behaviour. To address this, we conducted a systematic bioinformatics and experimental analysis, combining synthetic and natural peptides with studies on the RNA-binding protein FUS as a model system. Our results reveal that the sequence composition of RG/RGG motifs is a key determinant of their capacity for RNA-mediated condensate formation, stress granule recruitment, and transportin-1-mediated chaperoning and nuclear import. These findings provide new insight into the sequence grammar of disordered RG/RGG regions and how it encodes the multifunctionality of these proteins in cellular regulation.

molecular biology↗

bioRxiv2025

Molecular determinants of cardiac lymphatic dysfunction in a chronic pressure-overload model

Cardiac lymphatics have emerged as potential targets in cardiovascular diseases (CVDs). However, we recently reported that despite extensive lymphatic expansion during experimental cardiac pressure-overload, lymphatic drainage remained insufficient. To unravel the cellular and molecular mechanisms underlying lymphatic dysfunction in CVDs, we applied cardiac single-cell (sc) analyses in a murine heart failure model. Transaortic constriction (TAC), in C57BL/6J and BALB/c mice, was used to model chronic pressure-overload-induced cardiac hypertrophy and heart failure, respectively. Cardiac lymphatic (LEC) and blood vascular (BECs) endothelial cells were analyzed by scRNAseq (10XGenomics). Lymphatic targets were validated by immunohistochemistry and wholemount-imaging, and in vitro using human LEC cultures. We identified three distinct cardiac lymphatic subpopulations, capillary (LEC1), precollector (LEC2), and valvular (LEC3) clusters, and several BECs clusters, including venous BEC (vBEC). Chronic pressure-overload led to expansion of lymphatic capillaries and loss of valves in BALB/c, but not C75BL6/J. Analysis of differentially expressed genes (DEG) post-TAC revealed reduction only in BALB/c of lymphatic cell-junction components. In contrast, LEC expression of anchoring filaments, immune cell-adhesion molecules, and chemokines was preserved, or increased, indicating functional lymphatic-mediated immune cell uptake post-TAC. Interestingly, around 35% of DEGs identified in cardiac LECs post-TAC were similarly altered in interleukin (IL)-1{beta}-stimulated human LECs. In conclusion, loss of lymphatic valves and dysregulated lymphatic barrier properties may underly poor drainage capacity during pressure-overload, despite potent lymphangiogenesis and preserved LEC immune attraction. Further studies are needed to address how to restore lymphatic health to accelerate resolution of both inflammation and edema in CVDs.

cell biology↗

bioRxiv2024

Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

Despite recent progress in the ability to manipulate the genomes of eukaryotic cells1-3, there is still no effective and practical method to precisely integrate large synthetic DNA constructs into desired chromosomal sites using a programmable integrase. Serine integrases can perform the necessary molecular steps4, but only if their natural target site is first installed into the recipient genome by other methods. A more elegant approach would be to directly reprogram the serine integrase itself to target a desired genomic site that is different from the natural recognition site of the integrase5. Here, we describe the development of a Modular Integrase (MINT) platform, a versatile protein-guided genome editing tool that can facilitate site-directed targeted integration (TI) of synthetic DNA into chromosomal sites. Through a combination of structural modeling, directed evolution, and screening in human cells we have reprogrammed the specificity of the serine integrase Bxb1. We demonstrate the therapeutic potential of the MINT platform by retargeting Bxb1 to the human AAVS1 and TRAC loci where wild-type Bxb1 has no detectable activity. By combining the MINT platform with known activity-increasing Bxb1 mutants, we achieved 14% TI at the AAVS1 locus, and by additionally fusing zinc finger DNA binding domains to engineered Bxb1 variants, we achieved 35% TI at the TRAC locus in human K562 cells. To further demonstrate clinical potential, we achieved stable 15% TI of a functional donor construct in human T cells.

synthetic biology↗

bioRxiv2024

Vinegar injections can be used safely to control outbreaks of crown-of-thorns starfish (Acanthaster solaris) during the peak spawning season

Concerns are mounting over the effects of COTS outbreaks, prompting the need for integrated management strategies. Although direct control methods are short-term and localized, they remain one of the few operational tools that can be easily implemented by local actors. Vinegar injections have recently emerged as highly effective method; however their impact on reproductive behavior remains untested. Here, we investigated the short-term spawning response of mature COTS to double injections of household vinegar. First, COTS abundances and reproductive status were monitored during a massive outbreak affecting New Caledonias reefs. In-situ and laboratory experiments were then conducted to determine whether injected COTS would eventually release their eggs and trigger synchronized spawning among mature individuals in close proximity. Our results indicated that injections had no significant effect on spawning behavior, even in densely populated aggregations (>4000 COTS.ha-1). In the field, starfish exhibited ripe gonads with high gamete content (up to 35% of body weight) three days after conspecifics were injected. In the laboratory, mature COTS kept with injected, decaying individuals in a confined volume did not expel their gametes after two days. This suggests that vinegar injections could be used at any time, even during peak spawning, without risking synchronized spawning in the affected areas.

ecology↗

bioRxiv2023

Hexokinase 1 forms rings that constrict mitochondria during energy stress

Metabolic enzymes can adapt during energy stress, but the precise mechanisms and consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, clusters into ring-like structures around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial constriction sites. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the clustering of HK1. Moreover, we found several mutations that are critical for the formation of HK1-rings. Utilizing these mutations, we could show that HK1-rings keep mitochondria connected and rewire cellular metabolism during energy stress. Our findings highlight that HK1 is a robust energy stress sensor that regulates the shape, connectivity and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy stress-related pathologies.

cell biology↗