Béatrice Vallée - Academia.edu (original) (raw)

Papers by Béatrice Vallée

Research paper thumbnail of Streamlining N-terminally anchored yeast surface display via structural insights into S. cerevisiae Pir proteins

Microbial Cell Factories

Surface display co-opts yeast’s innate ability to embellish its cell wall with mannoproteins, thu... more Surface display co-opts yeast’s innate ability to embellish its cell wall with mannoproteins, thus converting the yeast’s 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 isoform’s N-terminus, e.g., through a short GPI-independent protein anchor. 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 β-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 ...

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

bioRxiv (Cold Spring Harbor Laboratory), Apr 4, 2022

While copper is an essential micronutrient and a technologically indispensable heavy metal, it is... more 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 lowthroughput 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. cerevisiae's 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 system's 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.

Research paper thumbnail of Neurofibromin, a new SUMO target involved in neurofibromatosis type 1 disease

HAL (Le Centre pour la Communication Scientifique Directe), Sep 13, 2019

Research paper thumbnail of Development of a bio­active fluorescent inhibitor probe targeting the Rho kinase ROCK for monitoring inhibition action

HAL (Le Centre pour la Communication Scientifique Directe), Jul 1, 2021

Research paper thumbnail of LIM Kinases: new anticancer therapeutic targets

HAL (Le Centre pour la Communication Scientifique Directe), Jun 2, 2017

Research paper thumbnail of New insights into the regulation of actin cytoskeleton dynamics via the Rho/ROCK/LIMK2/cofilin signalling pathway: a novel mechanism of regulation of cofilin by LIMK2”

HAL (Le Centre pour la Communication Scientifique Directe), Oct 7, 2021

Research paper thumbnail of Design, synthesis and biological evaluation of LIMK inhibitors

Research paper thumbnail of LIMK2-1, a new isoform of human LIMK2, regulates actin cytoskeleton remodeling via a different signaling pathway than that of its two homologs, LIMK2a and LIMK2b

HAL (Le Centre pour la Communication Scientifique Directe), Dec 6, 2018

LIMK2-1, a new isoform of Human LIMK2, regulates actin cytoskeleton remodeling via a different si... more LIMK2-1, a new isoform of Human LIMK2, regulates actin cytoskeleton remodeling via a different signaling pathway than that of its two homologs, LIMK2a and LIMK2b

Research paper thumbnail of High Efficiency of Histidylated-based Lipid Formulations for mRNA Transfection of Human Schwann Cells

HAL (Le Centre pour la Communication Scientifique Directe), Dec 6, 2021

Research paper thumbnail of Roles of LIM kinases in central nervous system function and dysfunction

FEBS letters, Jan 3, 2015

LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) regulate actin dynamics by phosphorylating cofilin.... more LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) regulate actin dynamics by phosphorylating cofilin. In this review, we outline studies that have shown an involvement of LIMKs in neuronal function and we detail some of the pathways and molecular mechanisms involving LIMKs in neurodevelopment and synaptic plasticity. We also review the involvement of LIMKs in neuronal diseases and emphasize the differences in the regulation of LIMKs expression and mode of action. We finally present the existence of a cofilin-independent pathway also involved in neuronal function. A better understanding of the differences between both LIMKs and of the precise molecular mechanisms involved in their mode of action and regulation is now required to improve our understanding of the physiopathology of the neuronal diseases associated with LIMKs.

Research paper thumbnail of New molecular insights into the crosstalk between Rho/ROCK/LIMK2/cofilin signalling pathway and Nf1

HAL (Le Centre pour la Communication Scientifique Directe), Jun 14, 2021

Research paper thumbnail of LIM Kinases: From Molecular to Pathological Features

Cells

LIM kinases (LIMKs), LIMK1 and LIMK2, are atypical kinases, as they are the only two members of t... more LIM kinases (LIMKs), LIMK1 and LIMK2, are atypical kinases, as they are the only two members of the LIM kinase family harbouring two LIM domains at their N-terminus and a kinase domain at their C-terminus [...]

Research paper thumbnail of Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Journal of Visualized Experiments

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many p... more Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death. In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by γ[ 32 P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

Research paper thumbnail of LIM Kinases, LIMK1 and LIMK2, Are Crucial Node Actors of the Cell Fate: Molecular to Pathological Features

Cells

LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the o... more LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the only two members of the LIM kinase family. They play a crucial role in the regulation of cytoskeleton dynamics by controlling actin filaments and microtubule turnover, especially through the phosphorylation of cofilin, an actin depolymerising factor. Thus, they are involved in many biological processes, such as cell cycle, cell migration, and neuronal differentiation. Consequently, they are also part of numerous pathological mechanisms, especially in cancer, where their involvement has been reported for a few years and has led to the development of a wide range of inhibitors. LIMK1 and LIMK2 are known to be part of the Rho family GTPase signal transduction pathways, but many more partners have been discovered over the decades, and both LIMKs are suspected to be part of an extended and various range of regulation pathways. In this review, we propose to consider the different molecular mech...

Research paper thumbnail of Confocal imaging of biomarkers at a single-cell resolution: quantifying 'living' in 3D-printable engineered living material based on Pluronic F-127 and yeast Saccharomyces cerevisiae

Biomaterials Research

Background Engineered living materials (ELMs) combine living cells with non-living scaffolds to o... more Background Engineered living materials (ELMs) combine living cells with non-living scaffolds to obtain life-like characteristics, such as biosensing, growth, and self-repair. Some ELMs can be 3D-printed and are called bioinks, and their scaffolds are mostly hydrogel-based. One such scaffold is polymer Pluronic F127, a liquid at 4 °C but a biocompatible hydrogel at room temperature. In such thermally-reversible hydrogel, the microorganism-hydrogel interactions remain uncharacterized, making truly durable 3D-bioprinted ELMs elusive. Methods We demonstrate the methodology to assess cell-scaffold interactions by characterizing intact alive yeast cells in cross-linked F127-based hydrogels, using genetically encoded ratiometric biosensors to measure intracellular ATP and cytosolic pH at a single-cell level through confocal imaging. Results When embedded in hydrogel, cells were ATP-rich, in exponential or stationary phase, and assembled into microcolonies, which sometimes merged into large...

Research paper thumbnail of Molecular mechanisms of the inhibition of the Rho/ROCK/LIMK2/cofilin pathway by the SecPH domain of Neurofibromin, Nf1, the protein responsible for Neurofibromatosis type I

Le Centre pour la Communication Scientifique Directe - HAL - Université de Nantes, Jul 3, 2021

Research paper thumbnail of Stressed by hydrogel? Physiological response of yeast cells embedded in thermoresponsive Pluronic F127: a prerequisite for bioreactor development

Le Centre pour la Communication Scientifique Directe - HAL - Diderot, Jul 3, 2021

Research paper thumbnail of Conception, synthèse et évaluation biologique d’inhibiteurs de LIM kinases

Research paper thumbnail of LIM kinases, new therapeutic targets to treat cancers, neurological disorders and Neurofibromatosis: development of small molecule inhibitors

Le Centre pour la Communication Scientifique Directe - HAL - memSIC, Jul 6, 2019

International audienc

Research paper thumbnail of Détermination et mise en œuvre d’un modèle in vivo pour tester de nouveaux inhibiteurs originaux des LIM kinases, cibles thérapeutiques émergentes dans le cancer

Le Centre pour la Communication Scientifique Directe - HAL - Diderot, May 23, 2019

International audienc

Research paper thumbnail of Streamlining N-terminally anchored yeast surface display via structural insights into S. cerevisiae Pir proteins

Microbial Cell Factories

Surface display co-opts yeast’s innate ability to embellish its cell wall with mannoproteins, thu... more Surface display co-opts yeast’s innate ability to embellish its cell wall with mannoproteins, thus converting the yeast’s 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 isoform’s N-terminus, e.g., through a short GPI-independent protein anchor. 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 β-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 ...

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

bioRxiv (Cold Spring Harbor Laboratory), Apr 4, 2022

While copper is an essential micronutrient and a technologically indispensable heavy metal, it is... more 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 lowthroughput 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. cerevisiae's 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 system's 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.

Research paper thumbnail of Neurofibromin, a new SUMO target involved in neurofibromatosis type 1 disease

HAL (Le Centre pour la Communication Scientifique Directe), Sep 13, 2019

Research paper thumbnail of Development of a bio­active fluorescent inhibitor probe targeting the Rho kinase ROCK for monitoring inhibition action

HAL (Le Centre pour la Communication Scientifique Directe), Jul 1, 2021

Research paper thumbnail of LIM Kinases: new anticancer therapeutic targets

HAL (Le Centre pour la Communication Scientifique Directe), Jun 2, 2017

Research paper thumbnail of New insights into the regulation of actin cytoskeleton dynamics via the Rho/ROCK/LIMK2/cofilin signalling pathway: a novel mechanism of regulation of cofilin by LIMK2”

HAL (Le Centre pour la Communication Scientifique Directe), Oct 7, 2021

Research paper thumbnail of Design, synthesis and biological evaluation of LIMK inhibitors

Research paper thumbnail of LIMK2-1, a new isoform of human LIMK2, regulates actin cytoskeleton remodeling via a different signaling pathway than that of its two homologs, LIMK2a and LIMK2b

HAL (Le Centre pour la Communication Scientifique Directe), Dec 6, 2018

LIMK2-1, a new isoform of Human LIMK2, regulates actin cytoskeleton remodeling via a different si... more LIMK2-1, a new isoform of Human LIMK2, regulates actin cytoskeleton remodeling via a different signaling pathway than that of its two homologs, LIMK2a and LIMK2b

Research paper thumbnail of High Efficiency of Histidylated-based Lipid Formulations for mRNA Transfection of Human Schwann Cells

HAL (Le Centre pour la Communication Scientifique Directe), Dec 6, 2021

Research paper thumbnail of Roles of LIM kinases in central nervous system function and dysfunction

FEBS letters, Jan 3, 2015

LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) regulate actin dynamics by phosphorylating cofilin.... more LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) regulate actin dynamics by phosphorylating cofilin. In this review, we outline studies that have shown an involvement of LIMKs in neuronal function and we detail some of the pathways and molecular mechanisms involving LIMKs in neurodevelopment and synaptic plasticity. We also review the involvement of LIMKs in neuronal diseases and emphasize the differences in the regulation of LIMKs expression and mode of action. We finally present the existence of a cofilin-independent pathway also involved in neuronal function. A better understanding of the differences between both LIMKs and of the precise molecular mechanisms involved in their mode of action and regulation is now required to improve our understanding of the physiopathology of the neuronal diseases associated with LIMKs.

Research paper thumbnail of New molecular insights into the crosstalk between Rho/ROCK/LIMK2/cofilin signalling pathway and Nf1

HAL (Le Centre pour la Communication Scientifique Directe), Jun 14, 2021

Research paper thumbnail of LIM Kinases: From Molecular to Pathological Features

Cells

LIM kinases (LIMKs), LIMK1 and LIMK2, are atypical kinases, as they are the only two members of t... more LIM kinases (LIMKs), LIMK1 and LIMK2, are atypical kinases, as they are the only two members of the LIM kinase family harbouring two LIM domains at their N-terminus and a kinase domain at their C-terminus [...]

Research paper thumbnail of Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Journal of Visualized Experiments

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many p... more Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death. In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by γ[ 32 P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

Research paper thumbnail of LIM Kinases, LIMK1 and LIMK2, Are Crucial Node Actors of the Cell Fate: Molecular to Pathological Features

Cells

LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the o... more LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the only two members of the LIM kinase family. They play a crucial role in the regulation of cytoskeleton dynamics by controlling actin filaments and microtubule turnover, especially through the phosphorylation of cofilin, an actin depolymerising factor. Thus, they are involved in many biological processes, such as cell cycle, cell migration, and neuronal differentiation. Consequently, they are also part of numerous pathological mechanisms, especially in cancer, where their involvement has been reported for a few years and has led to the development of a wide range of inhibitors. LIMK1 and LIMK2 are known to be part of the Rho family GTPase signal transduction pathways, but many more partners have been discovered over the decades, and both LIMKs are suspected to be part of an extended and various range of regulation pathways. In this review, we propose to consider the different molecular mech...

Research paper thumbnail of Confocal imaging of biomarkers at a single-cell resolution: quantifying 'living' in 3D-printable engineered living material based on Pluronic F-127 and yeast Saccharomyces cerevisiae

Biomaterials Research

Background Engineered living materials (ELMs) combine living cells with non-living scaffolds to o... more Background Engineered living materials (ELMs) combine living cells with non-living scaffolds to obtain life-like characteristics, such as biosensing, growth, and self-repair. Some ELMs can be 3D-printed and are called bioinks, and their scaffolds are mostly hydrogel-based. One such scaffold is polymer Pluronic F127, a liquid at 4 °C but a biocompatible hydrogel at room temperature. In such thermally-reversible hydrogel, the microorganism-hydrogel interactions remain uncharacterized, making truly durable 3D-bioprinted ELMs elusive. Methods We demonstrate the methodology to assess cell-scaffold interactions by characterizing intact alive yeast cells in cross-linked F127-based hydrogels, using genetically encoded ratiometric biosensors to measure intracellular ATP and cytosolic pH at a single-cell level through confocal imaging. Results When embedded in hydrogel, cells were ATP-rich, in exponential or stationary phase, and assembled into microcolonies, which sometimes merged into large...

Research paper thumbnail of Molecular mechanisms of the inhibition of the Rho/ROCK/LIMK2/cofilin pathway by the SecPH domain of Neurofibromin, Nf1, the protein responsible for Neurofibromatosis type I

Le Centre pour la Communication Scientifique Directe - HAL - Université de Nantes, Jul 3, 2021

Research paper thumbnail of Stressed by hydrogel? Physiological response of yeast cells embedded in thermoresponsive Pluronic F127: a prerequisite for bioreactor development

Le Centre pour la Communication Scientifique Directe - HAL - Diderot, Jul 3, 2021

Research paper thumbnail of Conception, synthèse et évaluation biologique d’inhibiteurs de LIM kinases

Research paper thumbnail of LIM kinases, new therapeutic targets to treat cancers, neurological disorders and Neurofibromatosis: development of small molecule inhibitors

Le Centre pour la Communication Scientifique Directe - HAL - memSIC, Jul 6, 2019

International audienc

Research paper thumbnail of Détermination et mise en œuvre d’un modèle in vivo pour tester de nouveaux inhibiteurs originaux des LIM kinases, cibles thérapeutiques émergentes dans le cancer

Le Centre pour la Communication Scientifique Directe - HAL - Diderot, May 23, 2019

International audienc