Biology subjects

Rebeaud, M. E.

Publications and source records attributed to Rebeaud, M. E..

7 recordsLinked to original sources

bioRxiv2025

Conserved intrinsically disordered region of DNAJB6 dictates its surveillance of FG-Nup condensates

Molecular chaperones are known for their role in preventing protein aggregation and assisting proteins in reaching their structurally functional state. DNAJB6, a J-domain protein that partners with Hsp70s and nucleotide exchange factors, is very potent in preventing amyloid formation of proteins with large intrinsically disordered regions (IDRs), including several disease-associated proteins. Complementary to this, we recently demonstrated a role for DNAJB6 in surveilling FG-Nucleoporins (FG-Nups) phase transitions and highlighted its role in nuclear pore complex assembly. We expand on this by showing that this activity of phase state surveillance is directed to several FG-Nups and shared with the closely related DNAJB2 and DNAJB8. We demonstrate that the surveillance mechanism of DNAJB6 is encoded in an unusually highly conserved IDR that promotes the formation of stable, gel-like assemblies of the chaperone itself. These assemblies likely provide a stable environment that can outcompete stable homotypic FG-Nup interactions and instead favors multivalent heterotypic chaperone:FG-Nup interactions. The evolutionary conservation of the DNAJB6-IDR, mutant analyses from both experimental in vitro and in cell data, and multiscale molecular dynamics simulations suggest that the sequence space for encoding stable gel-like assemblies is narrow and optimized to avoid self-aggregation while providing potent anti-amyloidogenic capacity.

biochemistry↗

bioRxiv2025

Subtle variations in a client protein determine bacterial Hsp90 dependence

Chaperones ensure protein homeostasis and are conserved across species. The ATP-dependent chaperone Hsp90 is present from bacteria to eukaryotes, where it stabilizes and activates a wide range of substrate proteins called clients. However, what determines whether a protein depends on Hsp90 remains an open question. Here, we focused on the bacterial chaperone Hsp90 and its obligate client TilS (referred to as TilSSo) in the bacterium Shewanella oneidensis. Although Hsp90 is indispensable in S. oneidensis under heat stress by protecting the essential protein TilSSo from degradation by the protease HslUV, Hsp90 is dispensable in Escherichia coli, suggesting that E. coli TilS (TilSEc) is Hsp90 independent. We therefore compared the TilS orthologs with respect to in vitro stability, in vivo degradation, and interaction with Hsp90 to identify the determinants of the Hsp90 dependence. We found that in contrast to TilSSo, TilSEc was more stable, was not degraded by protease in the absence of Hsp90, and did not interact with Hsp90, indicating that TilSEc is not a client of Hsp90. Chimeras between TilSSo and TilSEc as well as directed mutagenesis revealed a region of TilSSo that is key for protease degradation and Hsp90 protection. Consistent with these results, the growth of S. oneidensis producing TilSEc was no longer dependent on Hsp90 under heat stress. Conversely, Hsp90 became essential for the growth of E. coli that produced TilSSo instead of TilSEc. Taken together, these results provide new insights into the mechanism of client protection by Hsp90 and the interplay between chaperones and proteases.

microbiology↗

bioRxiv2025

On adaptation to a mesophilic environment and the chaperone network in Archaea

A prevailing hypothesis for the emergence of life on Earth holds that it might have originated in hydrothermal vents, where the environmental conditions, although physically and chemically extreme (acidity, lack of oxygen, high pressure, very high temperature), vary very little. According to this view, single-celled organisms appeared under these conditions subsequently began to colonize all aquatic environments, followed by terrestrial ones. Here, I study the proteomes of more than 250 reference proteomes of archaea as well as those of a few non-reference Promethearchaeati (ASGARD), which have an optimal growth temperature of between 10{degrees}C and 100{degrees}C. I found a correlation between the chaperome present in these organisms, and in particular the presence/absence of the HSP70 family (DnaK-DnaJ-GrpE, KJE for brevity) and the optimal growth temperature. These findings suggest that appearance of HSP70s in mesophilic living conditions was the key to greater adaptability of the organisms, to their ability to colonize different environments and, ultimately, led to the appearance of eukaryotes.

evolutionary biology↗

bioRxiv2025

Is Hsp110 boosting the basal disaggregation activity Hsp70 by enhanced entropic pulling strokes?

Hsp70s use energy from ATP hydrolysis to unfold protein structures and solubilize stable aggregates, accumulating native species even under adverse non-native conditions. To carry out its catalytic polypeptide-unfolding activity, Hsp70 needs to reversibly interact with a J-domain (JDP) catalyst, a misfolded or alternatively folded polypeptide substrate and a Nucleotide Exchange Factor (NEF), which binds to the Nucleotide Binding Domain (NBD) of HSp70, accelerates ADP-release and allosterically controls the dissociation of the unfolded polypeptide product of the unfolding reaction. Yet, during the process of eukaryotisation, GrpE was lost from the cytosol, to be replaced by novel NEF proteins, among which the Hsp110 family stands out. Hsp110s belong to the Hsp70 superfamily, but the evolutionary steps that led from an ancestral Hsp70 unfoldase to a Hsp110 NEF of Hsp70s remain unsolved. Combining experiments using wild-type Sse1 (yeast Hsp110) and rationally designed mutants, we show that Hsp110 is likely built upon some of distinctive features already present in Hsp70 by repurposing them, rather than by inventing novel molecular properties. Taking all results together, we suggest a novel mechanism of action of Hsp110, whereby it is a NEF that also enhances the unfolding/disaggregating entropic pulling forces generated by Hsp70, by transiently increasing the chaperone effective volume.

biochemistry↗

bioRxiv2024

Single-molecule evidence of Entropic Pulling by Hsp70 chaperones

Hsp70 chaperones are central components of the cellular network that ensure the structural quality of proteins. Despite their crucial roles in processes as diverse as the prevention of protein aggregation and protein translocation into organelles, their molecular mechanism of action has remained a hotly debated issue. Due to a lack of suitable methods, no experimental data has directly proven any of the models that have been proposed (Power Stroke, Brownian Ratchet, and Entropic Pulling). Recently, nanopores have emerged as a powerful tool to analyze the function of motor enzymes, as well as protein-protein interactions. Here, we used an in vitro single-molecule nanopore to mimic in vivo translocation of proteins, and to investigate the molecular mechanism of Hsp70. Our experiments demonstrate that Hsp70s forcefully extract polypeptide substrates that are trapped inside the pore. The forces they exert are strong at the molecular level, being equivalent to 46 pN over distances of 1 nm, and depend on the size of Hsp70. These findings provide unambiguous evidence supporting the Entropic Pulling mechanism of action of Hsp70s, thus solving a long-standing debate, and proposing a potentially universal principle governing diverse cellular processes. In addition, these results emphasize the utility of biological nanopores for studying protein function at the single-molecule level.

biophysics↗

bioRxiv2024

Autorepression of Yeast Hsp70 co-chaperones by intramolecular interactions involving their J-domains.

The Hsp70 chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) co-evolved with Hsp70s to trigger ATP-hydrolysis and catalytically upload various substrate polypeptides in need to be structurally modified by the chaperone. Here, we measured the protein disaggregation and refolding activities of the main yeast cytosolic Hsp70, Ssa1, in the presence of its most abundant JDPs, Sis1 and Ydj1, and two swap mutants, in which the J-domains have been interchanged. The observed differences by which the four constructs differently cooperate with Ssa1 and cooperate with each other, as well as their observed intrinsic ability to bind misfolded substrates and trigger Ssa1s ATPase, indicates the presence of yet uncharacterized intra-molecular dynamic interactions between the J-domains and their remaining C-terminal domains. Taken together, the data suggest an auto-regulatory role to these intra-molecular interactions within both type A and B JDPs, which might have evolved to reduce energy-costly ATPase cycles by the Ssa1-4 chaperones that are the most abundant Hsp70s in the yeast cytosol. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/578849v1_figa1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@d895d3org.highwire.dtl.DTLVardef@1545221org.highwire.dtl.DTLVardef@dc08b3org.highwire.dtl.DTLVardef@1bf286a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Lower panel: autoinhibited DnaJA or DnaJB dimers, drawn here as Swiss army knives with sequestered J-domains as folded blades, can bind misfolded polypeptides (violet). Upper panel: DnaJA or DnaJB become active when their J-domains are exposed and can bind ATP-Hsp70s, and transfer the misfolded polypeptides, respectively, onto Hsp70s nucleotide binding (Cyan) and protein binding domains (Orange and Green). Hsp70s interdomain linker (DLLLLDV, Magenta). C_FIG

biochemistry↗

bioRxiv2023

Design of an Arabidopsis thaliana reporter line to detect heat-sensing and signaling mutants

BackgroundGlobal warming is a major challenge for plant survival and growth. Understanding the molecular mechanisms by which higher plants sense and adapt to upsurges in the ambient temperature, is essential for developing strategies to enhance plant tolerance to heat stress. Here, we designed a special heat-responsive Arabidopsis thaliana reporter line that allowed an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature. MethodsA transgenic Arabidopsis thaliana reporter line named "Heat-Inducible Bioluminescence And Toxicity" (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase and D-amino acid oxidase, whose expression was found to be toxic only in the presence of D-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence of D-valine and analyzed for survival rate, bioluminescence and HSP gene expression. ResultsWhereas at 22{degrees}C, HIBAT seedlings grew unaffected by D-valine, and all survived following iterative heat treatments without D-valine, 98% died following heat treatments on D-valine. The HSP17.3B promoter was highly specific to heat, as it remained unresponsive to various plant hormones, Flagellin, H2O2, osmotic stress and high salt. Confirming that HIBAT does not significantly differ from its Col-0 parent, RNAseq analysis of heat-treated seedlings showed a strong correlation between the two lines. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at low, non-heat-shock temperatures. ConclusionThis study adds insights into the molecular mechanisms by which higher plants sense and adapt to rapid elevations of ambient temperatures. HIBAT was a valuable tool to identify Arabidopsis mutants defective in the response to high temperature stress. Our findings open new avenues for future research on the regulation of HSP expression and understanding their role in the onset of plant acquired thermotolerance.

plant biology↗