Biology subjects

Vallee, B.

Publications and source records attributed to Vallee, B..

6 recordsLinked to original sources

bioRxiv2026

Salt-enhanced surface display in the halotolerant yeast Debaryomyces hansenii

The halo-, xero-, and osmotolerant oleaginous yeast Debaryomyces hansenii can grow in extreme environments, with some isolates tolerating up to 4 M NaCl. As such, it could serve as a robust microbial chassis for non-sterile biotechnological processes based on seawater or saline side streams. However, its biotechnological toolbox lacks key genetic tools, particularly for inducible expression, secretion, and covalent anchoring of proteins to the outer cell surface. To address this gap, we characterised how growth in a medium supplemented with 1 M NaCl remodels the D. hansenii cell wall. We also developed strong constitutive, stationary-phase, and NaCl-inducible expression vectors, and designed protein tags for C- and N-terminal cell wall anchoring. These tools enabled functional surface display of {beta}-lactamase, acetylxylan esterase, and lipase A via native GPI-dependent and heterologous Pir-based anchors, while immunoblotting detected the reporters in both covalently and non-covalently bound cell wall fractions. Activities of surface-displayed enzymes increased by more than 80 % under hypersaline conditions, while DhCcw12-based C-terminal anchoring outperformed Pir-based N-terminal anchoring. Thus, we retooled the D. hansenii cell wall into a catalytically active biointerface and established this yeast as an osmotolerant chassis for hypersaline surface display.

molecular biology↗

bioRxiv2026

A new mechanism of regulation of LIM kinases, LIMK1 and LIMK2, modulates their activity on cofilin and actin filament remodelling

LIM kinases, LIMK1 and LIMK2, play a crucial role in cytoskeleton dynamics. They are involved in many physiological processes but also in several pathologies such as cancer, neuronal diseases and neurofibromatosis. Although LIM kinases appear as promising therapeutic targets, they remain undruggable. A better understanding of their activity and regulation is thus required to better design efficient targeted therapies. Here, we have shown the impact of a single amino acid on LIMK activity on cofilin, their main substrate in actin filament remodelling. We demonstrated that Y632 and Y630, for LIMK1 and LIMK2 respectively, mediate LIMK dimerization, resulting in their transphosphorylation. This process seems to be a prerequisite for their canonical phosphorylation on their respective T508 and T505 residues within the activation loop. These Tyrosine are not phosphorylated, their aromatic nature is rather critical to ensure proper LIMK activity on cofilin. These results bring new insights into LIMK molecular features.

Molecular Biology↗

bioRxiv2025

QUANTITATIVE PROTEOMICS OF PLASMA EXTRACELLULAR VESICLES REVEALS A TTR - PLASMINOGEN NETWORK IN ATTR CARDIAC AMYLOIDOSIS

BackgroundDespite recent progress, the prognosis of patients with transthyretin (TTR) cardiac amyloidosis remains poor; this is primarily due to late diagnosis, when irreversible damage has already occurred. Todays diagnostic work-up still relies on peripheral tissue or a cardiac biopsy, while circulating levels of TTR or other plasma markers have little diagnostic value. Although extracellular vesicles (EVs, as key mediators of intercellular communication) may reflect disease-specific molecular changes, their protein cargo has not yet been explored in the context of TTR amyloidosis (ATTR) cardiomyopathy. ObjectivesTo characterize the plasma EV proteome in ATTR cardiomyopathy and identify potential biomarkers for pathophysiological pathways, diagnosis, or prognosis. MethodsWe performed mass-spectrometry-based, label-free, proteomic profiling of plasma EVs from 65 patients with hypertrophic cardiomyopathy due to TTR amyloidosis (the ATTR+ group, n=41) or non-amyloid cardiac disease (the ATTR- group, n=24). The groups were matched by age and sex. ResultsA distinct protein signature comprising 117 deregulated proteins was identified in EVs from ATTR+ patients. The ATTR+ EVs were enriched in proteins associated with vascular homeostasis, coagulation, and inflammation. At least 18 of these proteins formed an interconnected network centered on plasmin/plasminogen. Notably, EV levels of TTR and plasminogen levels were elevated, while the level of alpha2-antiplasmin (plasmins primary inhibitor) was low. This imbalance is particularly relevant because plasmin is known to promote amyloidogenesis via TTR cleavage. ConclusionsOur findings provide new insights into the molecular mechanisms underlying ATTR cardiomyopathy and suggest that plasma EV proteins are potential diagnostic or prognostic biomarkers and/or therapeutic targets. CONDENSED ABSTRACTTTR amyloidosis (ATTR) causes severe cardiac damage, which is often diagnosed late. Through a comparative proteomic analysis of plasma extracellular vesicles (EVs) in patients with ATTR cardiomyopathy vs. patients with other cardiomyopathies, we identified several proteins of relevance to the pathophysiology of ATTR. Our analysis is the first to have highlighted an enrichment of plasmin/plasminogen (known to initiate the amyloidogenic process) and TTR in circulating EVs. Our results might foster the development of (i) diagnostic and prognostic markers for ATTR cardiomyopathy that do not require invasive procedures, and (ii) new therapeutic strategies. CENTRAL ILLUSTRATION - GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/662124v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@e64a55org.highwire.dtl.DTLVardef@11a1f38org.highwire.dtl.DTLVardef@1f7b1cborg.highwire.dtl.DTLVardef@b990aa_HPS_FORMAT_FIGEXP M_FIG C_FIG ETHICAL APPROVALThe present analysis was based on blood samples collected as part of a research project entitled "Study of the myocardial microenvironment and toxicity of amyloid proteins in patients with cardiac amyloidosis", which was approved by an institutional review board (CPP Sud- Mediterranee II, Marseille, France; approval references: 2021T2-12/2021-A00950-41 and 2022-A02416-37).

biochemistry↗

bioRxiv2023

Boosting N-terminally anchored yeast surface display via structural insights into S. cerevisiae Pir proteins

Surface display co-opts yeasts innate ability to embellish its cell wall with mannoproteins, thus converting the yeasts outer surface into a growing and self-sustaining catalyst. However, the efficient toolbox for converting the enzyme of interest into its surface-displayed isoform is currently lacking, especially if the isoform needs to be anchored to the cell wall near the isoforms N-terminus. Aiming to advance such N-terminally anchored surface display, we employed in silico and machine-learning strategies to study the 3D structure, function, genomic organisation, and evolution of the Pir protein family, whose members evolved to covalently attach themselves near their N-terminus to the {beta}-1,3-glucan of the cell wall. Through the newly-gained insights, we rationally engineered 14 S. cerevisiae Hsp150 (Pir2)-based fusion proteins. We quantified their performance, uncovering guidelines for efficient yeast surface display while developing a construct that promoted a 2.5-fold more efficient display than the full-length Hsp150 and a Pir-tag, i.e., a peptide spanning only 4.5 kDa but promoting as efficient surface display as the full-length Hsp150. These constructs fortify the existing surface display toolbox, allowing for a prompt and routine refitting of any protein into its N-terminally anchored isoform. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/538238v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@909e7aorg.highwire.dtl.DTLVardef@9480ceorg.highwire.dtl.DTLVardef@1945bdcorg.highwire.dtl.DTLVardef@11ae505_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Re-engineering of CUP1 promoter and Cup2/Ace1 transactivator to convert Saccharomyces cerevisiae into a whole-cell eukaryotic biosensor capable of detecting 10 nM of bioavailable copper

While copper is an essential micronutrient and a technologically indispensable heavy metal, it is toxic at high concentrations, harming the environment and human health. Currently, copper is monitored with costly and low-throughput analytical techniques that do not evaluate bioavailability, a crucial parameter which can be measured only with living cells. We overcame these limitations by building upon yeast S. cerevisiaes native copper response and constructed a promising next-generation eukaryotic whole-cell copper biosensor. We combined a dual-reporter fluorescent system with an engineered CUP1 promoter and overexpressed Cup2 transactivator, constructing through four iterations a total of 16 variants of the biosensor, with the best one exhibiting a linear range of 10-8 to 10-3 M of bioavailable copper. Moreover, this variant distinguishes itself by superior specificity, detection limit, and linear range, compared to other currently reported eukaryotic and prokaryotic whole-cell copper biosensors. By re-engineering the transactivator, we altered the systems sensitivity and growth rate, while assessing the performance of Cup2 with heterologous activation domains. Thus, in addition to presenting the next-generation whole-cell copper biosensor, this work urges for an iterative design of eukaryotic biosensors and paves the way toward higher sensitivity through transactivator engineering. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2021

Noncanonical structural requirements of neurofibromin SUMOylation reveal a folding-deficiency of several pathogenic mutants

Neurofibromin (Nf1) is a large multidomain protein encoded by the tumour-suppressor gene NF1. NF1 is mutated in a frequently occurring genetic disease, neurofibromatosis type I, and in various cancers. The best described function of Nf1 is its Ras-GTPase activity, carried out by its GAP-related domain (GRD). SecPH, another structurally well-characterized domain of Nf1, is immediately adjacent to the GRD and interacts with lipids and proteins, thus connecting Nf1 to diverse signalling pathways. Here, we demonstrate, for the first time, that Nf1 and SecPH are substrates of the SUMO pathway. We identified a well-defined SUMOylation profile of SecPH and a main SUMOylation event on Lys1731 that appears to play a role in Ras-GAP activity. Our data allowed us to characterize a new set of pathogenic Nf1 missense mutants that exhibits a disrupted SUMOylation profile that may correlate with their unfolding. Accordingly, Lys1731 SUMOylation is mediated by a noncanonical structural motif, therefore allowing a read-out of SecPH conformation and folding status.

molecular biology↗