Biology subjects

Lemieux, P.

Publications and source records attributed to Lemieux, P..

6 recordsLinked to original sources

bioRxiv2026

The hydrophobicity of peptides limits their availability to bind to interacting domains

A challenge in synthetic biology is to transfer the knowledge acquired in vitro to in vivo applications. For instance, binding affinity between peptide binding domains (PBDs) and their binding peptides is usually measured in simple buffers and in the absence of the cell machinery responsible for synthesis and proteostasis. Consequently, there is often a discrepancy between interaction quantified in vitro and in vivo. To identify strongly binding PBD-peptide pairs for in vivo applications, we aim to better understand the determinants of synthetic interaction in living yeast cells. We designed libraries of peptides expected to bind to PBDs based on data collected by phage-display. Using an in vivo protein interaction assay, we assessed the interaction strength of >6,500 PBD-peptide variants in yeast. We found that most substitutions disrupt binding, but that 3.4% increased binding strength to levels higher than expected from the phage-display data. We asked if, by using our mutational data, it was possible to optimize peptide sequences to gain even more binding strength and show additivity effect up to four substitutions. We found that PBD specificity is consistent between in vitro and in vivo assays. We hypothesized that in vivo peptide abundance could contribute to the discrepancy between the PCA and the phage-display results. We performed assays to detect in vivo peptide availability, reporting on peptide abundance, and we found that peptides with a stronger binding also show a higher availability in cells. We show that peptide availability, which seems mainly defined by peptide hydrophobicity, is key for the design of strong synthetic interactions.

molecular biology↗

bioRxiv2025

A protein-fragment complementation assay to quantify synthetic protein scaffold efficiency

Scaffolds are powerful tools in synthetic biology used for various applications, from increasing yield to optimizing signalling specificity. Protein scaffolds can be built by fusing peptide binding domains (PBD) and attaching the peptide they bind to the enzymes, inducing spatial proximity. Only a few PBD-peptide combinations have been tested in this context, and no combination produced a high yield in yeast, an important chassis in biotechnology. Therefore, there is a need for more exploration of PBD-peptide pairs to be used in this model. Scaffold characterization is challenging because it is often dependent on a model pathway with an output that is difficult to measure quantitatively. Here, we use a protein-fragment complementation assay (PCA) to study scaffolding efficiency in yeast, which allows to couple scaffolding efficiency with growth rate. First, we characterize the strength of PBD-peptide interactions (PPI) and the binding availability of the PBDs and peptides. Then, we test different scaffold architectures and expression levels to quantify the simultaneous binding of peptide pairs to the scaffold. We show that PPI strength of the weakest binding PBD-peptide pair is critical for scaffolding efficiency and that PPI strength is limited by low binding availability of some domains and peptides in vivo. Also, we find that slight architectural variations and expression levels have a significant impact on scaffolding efficiency detected by DHFR PCA. Finally, we used DHFR PCA approaches to characterize novel PBD-peptide pairs and we identified pairs to expand the sequence toolbox for scaffold design in yeast through DHFR PCA easy-to-read signal.

synthetic biology↗

bioRxiv2024

Cryptic genetic variation shapes the fate of gene duplicates in a protein interaction network

Paralogous genes are often redundant for long periods of time before they diverge in function. While their functions are preserved, paralogous proteins can accumulate mutations that, through epistasis, could impact their fate in the future. By quantifying the impact of all single-amino acid substitutions on the binding of two myosin proteins to their interaction partners, we find that the future evolution of these proteins is highly contingent on their regulatory divergence and the mutations that have silently accumulated in their protein binding domains. Differences in the promoter strength of the two paralogs amplify the impact of mutations on binding in the lowly expressed one. While some mutations would be sufficient to non-functionalize one paralog, they would have minimal impact on the other. Our results reveal how functionally equivalent protein domains could be destined to specific fates by regulatory and cryptic coding sequence changes that currently have little to no functional impact.

evolutionary biology↗

bioRxiv2023

Dissection of the role of a SH3 domain in the evolution of binding preference of paralogous proteins

Protein-protein interactions (PPIs) drive many cellular processes. Some PPIs are directed by Src homology 3 (SH3) domains that bind proline-rich motifs on other proteins. The evolution of the binding specificity of SH3 domains is not completely understood, particularly following gene duplication. Paralogous genes accumulate mutations that can modify protein functions and, for SH3 domains, their binding preferences. Here, we examined how the binding of the SH3 domains of two paralogous yeast type I myosins, Myo3 and Myo5, evolved following duplication. We found that the paralogs have subtly different SH3-dependent interaction profiles. However, by swapping SH3 domains between the paralogs and by characterizing the SH3 domains freed from their protein context, we find that very few of the differences in interactions, if any, depend on the SH3 domains themselves. We used ancestral sequence reconstruction to resurrect the pre-duplication SH3 domains and examined, moving back in time, how the binding preference changed. Although the closest ancestor of the two domains had a very similar binding preference as the extant ones, older ancestral domains displayed a gradual loss of interaction with the modern interaction partners when inserted in the extant paralogs. Molecular docking and experimental characterization of the free ancestral domains showed that their affinity with the proline motifs is likely not the cause for this loss of binding. Taken together, our results suggest that the SH3 and its host protein could create intramolecular or allosteric interactions essential for the SH3-dependent PPIs, making domains not functionally equivalent even when they have the same binding specificity.

evolutionary biology↗

bioRxiv2022

Exit of spore dormancy transforms the yeast cytoplasm and the solubility of its proteome

The biophysical properties of the cytoplasm are major determinants of key cellular processes and adaptation. Many yeasts produce dormant spores that can withstand extreme conditions. We show that spores of Saccharomyces cerevisiae exhibit extraordinary biophysical properties, including a highly viscous and acidic cytosol. These conditions alter the solubility of more than 100 proteins such as metabolic enzymes that become more soluble as spores transit to active cell proliferation upon nutrient repletion. A key regulator of this transition is the heat shock protein Hsp42, which shows transient solubilization and phosphorylation, and is essential for the transformation of the cytoplasm during germination. Germinating spores therefore return to growth through the dissolution of protein assemblies, orchestrated in part by Hsp42 activity. The modulation of spores molecular properties are likely key adaptive features of their exceptional survival capacities.

cell biology↗

bioRxiv2021

Toeholder: a software for automated design and in silico validation of toehold riboswitches

Synthetic biology aims to engineer biological circuits, which often involve gene expression. A particularly promising group of regulatory elements are riboswitches because of their versatility with respect to their targets, but early synthetic designs were not as attractive because of a reduced dynamic range with respect to protein regulators. Only recently, the creation of toehold switches helped overcome this obstacle by also providing an unprecedented degree of orthogonality. However, a lack of automated design and optimization tools prevents the widespread and effective use of toehold switches in high throughput experiments. To address this, we developed Toeholder, a comprehensive open-source software for toehold design and in silico comparison. Toeholder takes into consideration sequence constraints from experimentally tested switches, as well as data derived from molecular dynamics simulations of a toehold switch. We describe the software and its in silico validation results, as well as its potential applications and impacts on the management and design of toehold switches.

bioinformatics↗