Biology subjects

Rebeaud, M. E.

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

3 recordsLinked to original sources

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↗