Biology subjects

Vallee, B.

Publications and source records attributed to Vallee, B..

3 recordsLinked to original sources

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↗