Biology subjects

Fauvet, B.

Publications and source records attributed to Fauvet, B..

5 recordsLinked to original sources

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

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↗

bioRxiv2022

A novel fluorescent multi-domain protein construct reveals the individual steps of the unfoldase action of Hsp70

A detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the detailed characterization of the aggregates, which are typically heterogeneous. To tackle this problem, we designed here a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase core, flanked by stress-resistant fluorescent mTFP and Venus domains, which upon denaturation formed a discrete stable population of small aggregates. Combining Forster Resonance Energy Transfer and enzymatic activity measurements provided unprecedent details on MLucV states, including native, aggregated, unfolded and chaperone-bound conformations. Using MLucV, we probed the various steps undertaken by bacterial Hsp70 to convert stable discrete aggregates into native proteins. The mechanism first involved an ATP-fuelled disaggregation and unfolding step of the stable pre-aggregated substrate, with a consequent stretching of MLucV beyond simply-unfolded conformations, followed, upon release, by native refolding. Furthermore, the ATP-fuelled unfolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures, highly adverse to the native state. These results unambiguously excluded binding and preventing aggregation from the non-equilibirum mechanism by which Hsp70 converts stable aggregates into metastable native proteins.

molecular biology↗

bioRxiv2018

Bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates preferentially by HslUV proteolysis

Whereas in eukaryotic cells, the Hsp90s are profusely-studied molecular chaperones controlling protein homeostasis together with Hsp70s, in bacteria, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains unknown. To uncover physiological processes depending on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild type E. coli, and from knockout mutants{Delta} dnaKdnaJ ({Delta}KJ),{Delta} htpG ({Delta}G) and{Delta} dnaKdnaJ{Delta}htpG ({Delta}KJG) and compared their growth rates under heat-stress also with{Delta} dnaKdnaJ{Delta}hslV. Whereas, expectedly, mutant {Delta}G showed no proteomic differences with wild-type, {Delta}KJ expressed more chaperones, proteases and ribosomes and dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that {Delta}KJG showed higher levels of metabolic and respiratory enzymes and both {Delta}KJG and{Delta} dnaKdnaJ{Delta}hslV grew better at 37oC than {Delta}KJ. The results indicate that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates, preferably by the HslUV protease.\n\nSignificance statementThe molecular chaperones Hsp70 and Hsp90 are among the most abundant and well-conserved proteins in all realms of life, forming together the core of the cellular proteostasis network. In eukaryotes, Hsp90 functions in collaboration with Hsp70; we studied this collaboration in E. coli, combining genetic studies with label-free quantitative proteomics in which both protein abundance and protein solubility were quantified. Bacteria lacking Hsp70 (DnaK) and its co-chaperone DnaJ ({Delta}dnaKdnaJ) grew slower and contained significantly less key metabolic and respiratory enzymes. Unexpectedly, an additional deletion of the Hsp90 (htpG) gene partially restored the WT phenotype. Deletion of the HslV protease in the {Delta}dnaKdnaJ background also improved growth, suggesting that bacterial Hsp90 mediates the degradation of Hsp70 substrates, preferentially through HslV.\n\nAt 37oC{Delta} dnaKdnaJ E. coli mutants grow slower than wild type cells. Quantitative proteomics shows that compared to wild type cells,{Delta} dnaKdnaJ cells grown at 30oC contain significantly less key metabolic and respiratory enzymes. Unexpectedly, deletion of the HtpG gene in the {Delta} dnaKdnaJ background ameliorates growth at 37oC and partially restores the cellular levels of some metabolic and respiratory enzymes.

molecular biology↗

bioRxiv2017

Molecular chaperones inject energy from ATP hydrolysis into the non-equilibrium stabilisation of native proteins

Protein homeostasis, namely the ensemble of cellular mechanisms collectively controlling the activity, stability and conformational states of proteins, depends on energy-consuming processes. De novo protein synthesis requires ATP hydrolysis for peptide bond formation. Controlled degradation by the chaperone-gated proteases requires ATP hydrolysis to unfold target proteins and render their peptide bonds accessible to hydrolysis. During and following translation, different classes of molecular chaperones require ATP hydrolysis to control the conformational state of proteins, favor their folding into their active conformation and avoid, under stress, their conversion into potentially harmful aggregates. Furthermore, specific ATP-fueled unfolding chaperones can dynamically revert aggregation itself. We used here various biochemical assays and physical modeling to show that both bacterial chaperones GroEL (HSP60) and DnaK (HSP70) can use the energy liberated by ATP hydrolysis to maintain proteins in their active state even under conditions that do not favor, thermodynamically, the native state. The energy from ATP hydrolysis is thus injected by the chaperones in the system and converted into an enhanced, non-equilibrium steady-state stabilization of the native state of their substrates. Upon ATP consumption, the chaperone substrates spontaneously revert to their equilibrium non-native state.

biochemistry↗