Bruno Fauvet

University of Lausanne | UNIL · Department of Plant Molecular Biology

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 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...

Detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the lack of a comprehensive characterization of the aggregates, which are typically heterogeneous. Here we designed a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase flanked by stress-resistant fluor...

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 stres...

Life is a non-equilibrium phenomenon. Owing to their high free energy content, the macromolecules of life tend to spontaneously react with ambient oxygen and water and turn into more stable inorganic molecules. A similar thermodynamic picture applies to the complex shapes of proteins: While a polypeptide is emerging unfolded from the ribosome, it m...

In eukaryotes, the 90-kDa heat shock proteins (Hsp90s) are profusely studied chaperones that, together with 70-kDa heat shock proteins (Hsp70s), control protein homeostasis. In bacteria, however, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains poorly characterized. To uncover physiological processes that depend on HtpG...

Erythropoiesis is a tightly regulated cell differentiation process in which specialized oxygen- and carbon dioxide-carrying red blood cells are generated in vertebrates. Extensive reorganization and depletion of the erythroblast proteome leading to the deterioration of general cellular protein quality control pathways and rapid hemoglobin biogenesi...

At dawn of a scorching summer day, land plants must anticipate upcoming extreme midday temperatures by timely establishing molecular defenses that can keep heat‐labile membranes and proteins functional. A gradual morning pre‐exposure to increasing sub‐damaging temperatures induces heat‐shock proteins (HSPs) that are central to the onset of plant ac...

Mortality in COVID-19 patients predominantly results from an acute respiratory distress syndrome (ARDS), in which lungs alveolar cells undergo programmed cell death. Mortality in a sepsis-induced ARDS rat model is reduced by adenovirus over-expression of the HSP70 chaperone. A natural rise of body temperature during mild fever can naturally accumul...

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 ana...

During and after protein translation, molecular chaperones require ATP hydrolysis to favor the native folding of their substrates and, under stress, to avoid aggregation and revert misfolding. Why do some chaperones need ATP, and what are the consequences of the energy contributed by the ATPase cycle? Here, we used biochemical assays and physical m...

Cadmium is a highly poisonous metal and a human carcinogen, but the molecular mechanisms underlying its cellular toxicity are not fully understood. Recent findings in yeast cells indicate that cadmium exerts its deleterious effects by inducing widespread misfolding and aggregation of nascent proteins. Here, we discuss this novel mode of toxic heavy...

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 t...

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age- associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mech...

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mecha...

Variation of aSyn acetylation levels with ageing. Brain protein extracts of WT and T2.KO young (2 month) and old (8 month) mice were probed for acetyl-lysine (red) and aSyn (green) (n = 4 per group). The ratio of acetyl-lysine/aSyn ratio is presented. **p < 0.01, **** p < 0.0001, ordinary one-way ANOVA followed by Tukey’s multiple comparisons test....

Acetylation-resistant aSyn mutant maintains the typical NMR signal and membrane-binding ability. (A) Superposition of 2D 1H-15N HSQC NMR spectra of recombinant 15N-labelled aSyn WT (black), K6+10R (red). (D) Residue-specific changes in 1H-15N HSQC signal intensities of aSyn WT (black) and aSyn K6+10R (red) upon addition of SUVs formed by POPC:POPA...

Acetylation-resistant mutant of aSyn is toxic. Lactate dehydrogenase levels (LDH) were measured in the supernatants of primary cultures infected with AVV6 encoding for WT, KQ or KR aSyn, at different time points after transduction. The KR mutant is toxic 3 days after infection, and the toxicity is then indistinguishable at later time points. Data i...

Expression of WT aSyn in the SN is toxic over time. (A) Brain sections immunostained for TH (red panels) and aSyn (Syn-1) (green panels) 1, 2 and 3 weeks after injection with vectors encoding for EGFP or WT aSyn. Scale bar for isolated channels 200 μm and for merged channels 100 μm. (B) Stereological counting of the number of TH-positive neurons in...

Aggregation pattern of aSyn in the rat substantia nigra. Brain sections immunostained for aggregated-aSyn (red) and GFP or pS129 aSyn (green) from representative animals 3 weeks after injection with AAV6 vectors encoding for GFP and WT, KR or KQ aSyn. (A) 5G4 and GFP (GFP group) or pS129 (aSyn groups) merged signal with DAPI is presented. Scale bar...

Raw data. Results from Figs 1F, 2B, 2F, 3A, 3B, 4A, 4B, 4C, 5C, 6B, 7B, 7E, 7F, S1, S4, S6 and S7B. (XLSX)

aSyn KxK motifs are less susceptible to chemical acetylation in vitro. Peptide mass spectrometry analysis of chemically acetylated recombinant aSyn, showing the number of acetylation occurrences. Each green bar represents a detected peptide, and a red dash indicates an acetylation. (TIF)

aSyn acetylation in mouse brain, identified by peptide mass fingerprint (PMF) (trypsin digestion). Theoretical peptide mass (Da); error (ppm); Start-end identified peptides; peptide sequences; putative acetylation residues. Oxid (M), N-terminal acetylation and acetylation (K) as variable modifications. (DOCX)

Autophagy is impaired upon T2.KD in the cell model of aSyn aggregation. Protein extracts from Scr or T2.KD cells co-transfected with SynT and Synphilin-1 were probed for P62 and β-Actin. Normalized levels of P62 are presented (n = 3). ** p < 0.01, unpaired t-test with equal SD. Data in S1 Data.xls. (TIF)

aSyn constructs, viral vector details, and experimental paradigm. (A) aSyn double mutants mimicking the acetylated (KQ) or the acetylation-resistant (KR) variants of aSyn on K6 and K10. (B) Recombinant adeno-associated viral vectors (AAV) serotype 6 expressing green fluorescent protein (GFP), human wild type (WT), KQ, KR variants of aSyn under the...

Alpha-synuclein is a presynaptic protein of poorly understood function that is linked to both genetic and sporadic forms of Parkinson's disease. We have proposed that alpha-synuclein may function specifically at synaptic vesicles docked at the plasma membrane, and that the broken-helix state of the protein, comprising two antiparallel membrane-boun...

Posttranslational modifications (PTMs) serve as molecular switches for regulating protein folding, function, and interactome and have been implicated in the misfolding and amyloid formation by several proteins linked to neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Understanding the role of individual PTMs in protein mi...

Over the last two decades, the identification of missense mutations in the α-synuclein (α-Syn) gene SNCA in families with inherited Parkinson disease (PD) has reinforced the central role of α-Syn in PD pathogenesis. Recently, a new missense mutation (H50Q) in α-Syn was described in patients with a familial form of PD and dementia. Here we investiga...

Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective...

Significance This study describes the nonenzymatic preparation of a protein conjugated to a tetraubiquitin Lys48-based chain made of native isopeptide bonds. We show that certain properties of our model protein, the Parkinson disease-associated protein α-Synuclein (α-Syn), are differentially regulated depending on the length of the conjugated ubiqu...

Post-translational modifications (PTMs) regulate key aspects of the physiological and pathogenic properties of Parkinson's disease-associated presynaptic protein α-synuclein. We herein describe a one-pot total chemical synthesis that should enable site-specific introduction of single or multiple PTMs or small molecule probes essentially at any site...

Developing novel therapies against neurodegenerative disorders requires the ability to detect their early, pre-symptomatic manifestations to enable treatment before irreversible cellular damage occurs. Precocious signs indicative of neurodegeneration include characteristic changes in certain protein levels, which can be used as diagnostic biomarker...

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of...

Sedimentation rates of wt and Leu46 mutants are independent of protein concentration, in the range tested (5–50 µM). C(s) distributions of wt huMIF (A), L46F huMIF (B), L46A huMIF (C) and L46G huMIF (d) at 50 µM (solid lines), 15 µM (dashed lines) and 5 µM (dotted lines). (TIFF)

NMR chemical shift measurements demonstrate a high similarity between L46A and L46G huMIF, whereas the L46F mutant exhibits a slightly different chemical shift pattern. (A) Two selected regions in 1H-15N HSQC spectra are shown for residues with strong chemical shift deviation. Color codes are as follows: wild-type in black, L46A mutant in red, L46F...

MIF Leishmania homologues adopt extended α-helix H1. (A) Superimposition of wt human and Leishmania MIF monomers. Note the extension of the helix H1 in the Leishmania species, in comparison to the crystal structure of the human protein. (B) Multiple sequence alignment of wt huMIF and the two species of Leishmania MIF. Residues highlighted in square...

Root mean square fluctuations (RMSF, a measure of the average atomic mobility) of the Cα atoms during the molecular dynamics simulations of wt and Leu46 mutants. Black line, wt huMIF; blue line, L46F huMIF; red line, L46A huMIF; green line, L46G huMIF. (TIFF)

Thermal denaturation of wt and mutant MIF is not concentration-dependant. Thermal unfolding studies of wt huMIF (A), L46F huMIF (B), 46A huMIF (C) and L46G huMIF (D) were monitored by far-UV CD at 218 nm. Proteins were prepared in PBS 1X, pH 7.4. Solid lines, 30 µM; dashed lines, 10 µM; dotted lines, 5 µM. (TIFF)

N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively N(α)-acetylated in vivo. Nevertheless, most of the biochemical an...

Despite increasing evidence that supports the role of different post-translational modifications (PTMs) in modulating α-synuclein (α-syn) aggregation and toxicity, relatively little is known about the functional consequences of each modification and whether or not these modifications are regulated by each other. This lack of knowledge arises primar...

It has been more than a century since the first evidence linking the process of amyloid formation to the pathogenesis of Alzheimer's disease. During the last three decades in particular, increasing evidence from various sources (pathology, genetics, cell culture studies, biochemistry, and biophysics) continues to point to a central role for the pat...

Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helica...

α-Synuclein (α-syn) is a 140-amino acid protein that plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. However, the molecular determinants that are responsible for triggering and/or propagating α-syn aggregation and toxicity remain poorly understood. Several studies have suggested that there are direc...

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase act...

Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities usin...

Cortical dynamics can be imaged at high spatiotemporal resolution with voltage-sensitive dyes (VSDs) and calcium-sensitive dyes (CaSDs). We combined these two imaging techniques using epifluorescence optics together with whole cell recordings to measure the spatiotemporal dynamics of activity in the mouse somatosensory barrel cortex in vitro and in...