A Novel View on the Role of Intracellular Tails in Surface Delivery of the Potassium-Chloride Cotransporter KCC2

A plethora of neurological disorders are associated with alterations in the expression and localization of potassium-chloride cotransporter type 2 (KCC2), making KCC2 a critical player in neuronal function and an attractive target for therapeutic treatment. The activity of KCC2 is determined primarily by the rates of its surface insertion and internalization. Currently the domains of KCC2 dictating its trafficking and endocytosis are unknown. Here, using live-cell immunolabeling and biotinylation of KCC2 proteins expressed in murine neuroblastoma N2a cells, human embryonic kidney 293 cells, or primary cultures of rat hippocampal neurons, we identified a novel role for the intracellular N and C termini in differentially regulating KCC2 surface expression. We report that the N terminus is required for KCC2 insertion into the plasma membrane, whereas the C terminus is critical for the membrane stability of KCC2. Our results provide novel insights into the structure–function role of specific KCC2 domains and open perspectives in exploring structural organization of this protein.Peer reviewe

ETUDE STRUCTURALE ET FONCTIONNELLE DE LA PROTEINE CHAPERON D'HISTONES ASF1

Eukaryotic genomic DNA is packed into chromatine as nucleosomes. This packing is essential for the DNA to fit inside the nucleus and for protecting DNA against oxydative aggressions. Additionally, the chromatine structure tightly regulates processes occurring on the DNA template. This work is dedicated to understanding the molecular mechanism underlying chromatin remodeling. Here we focus on the histones H3 and H4, two of the four nucleosome core particules, and the histone chaperone Asf1. We solved by NMR the structure of Asf1 in complex with the histones. This result constitute an essential step firstly to study in vivo the function of this interaction and secondly to initiate the design of specific peptidic inhibitors of this interaction which are also potential anticancer agents. We also developped an in vitro method to study the molecular basis of nucleosome assembly and disasssembly by Asf1.Au sein des cellules eucaryotes, l'ADN est compacté sous la forme de chromatine dont l'unité de base est le nucléosome. Cette structure protéique protège l'ADN des agressions oxydantes et a un rôle essentiel dans la régulation des processus nucléaires. Ce travail a pour objectif de mieux comprendre les mécanismes de remodelage de la chromatine. Nous nous sommes plus particulièrement intéressés à l'interaction entre les protéines du nucléosome que sont les histones H3 et H4 et le chaperon d'histones Asf1. Nous avons résolu par RMN la structure du complexe entre Asf1 et les histones. Ce travail a constitué une base pour, d'une part, analyser la fonction de cette interaction in vivo et, d'autre part, initier la conception de peptides inhibiteurs spécifiques de cette interaction et anti-tumoraux potentiels. Au cours de ce travail, nous avons également développé une méthode in vitro d'étude des mécanismes moléculaires d'assemblage et de désassemblage de nucléosomes par Asf1

Etude structurale et fonctionnelle de la protéine chaperon d'histones Asf1

Au sein des cellules eucaryotes, l ADN est compacté sous la forme de chromatine dont l unité de base est le nucléosome. Cette structure protéique protège l ADN des agressions oxydantes et a un rôle essentiel dans la régulation des processus nucléaires. Ce travail a pour objectif de mieux comprendre les mécanismes de remodelage de la chromatine. Nous nous sommes plus particulièrement intéressés à l interaction entre les protéines du nucléosome que sont les histones H3 et H4 et le chaperon d histones Asf1. Nous avons résolu par RMN la structure du complexe entre Asf1 et les histones. Ce travail a constitué une base pour, d une part, analyser la fonction de cette interaction in vivo et, d autre part, initier la conception de peptides inhibiteurs spécifiques de cette interaction et anti-tumoraux potentiels. Au cours de ce travail, nous avons également développé une méthode in vitro d étude des mécanismes moléculaires d assemblage et de désassemblage de nucléosomes par Asf1PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

In Vivo Study of the Nucleosome Assembly Functions of ASF1 Histone Chaperones in Human Cells▿ †

Histone chaperones have been implicated in nucleosome assembly and disassembly as well as histone modification. ASF1 is a highly conserved histone H3/H4 chaperone that synergizes in vitro with two other histone chaperones, chromatin assembly factor 1 (CAF-1) and histone repression A factor (HIRA), in DNA synthesis-coupled and DNA synthesis-independent nucleosome assembly. Here, we identify mutants of histones H3.1 and H3.3 that are unable to interact with human ASF1A and ASF1B isoforms but that are still competent to bind CAF-1 and HIRA, respectively. We show that these mutant histones are inefficiently deposited into chromatin in vivo. Furthermore, we found that both ASF1A and ASF1B participate in the DNA synthesis-independent deposition of H3.3 in HeLa cells, thus highlighting an unexpected role for ASF1B in this pathway. This pathway does not require interaction of ASF1 with HIRA. We provide the first direct determination that ASF1A and ASF1B play a role in the efficiency of nucleosome assembly in vivo in human cells

Footprinting of protein interactions by tritium labeling

International audienceA new footprinting method for mapping protein interactions has been developed, using tritium as a radioactive label. As residues involved in an interaction are less labeled when the complex is formed, they can be identified via comparison of the tritium incorporation of each residue of the bound protein with that of the unbound one. Application of this footprinting method to the complex formed by the histone H3 fragment H3122−135 and the protein hAsf1A1−156 afforded data in good agreement with NMR results

Quantification of Detergents Complexed with Membrane Proteins

International audienc

Quantification of Detergents Complexed with Membrane Proteins.

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies

Conception sur une base rationnelle de peptides de haute affinité inhibant l'histone chaperon ASF1

International audienceAnti-silencing function 1 (ASF1) is a conserved H3-H4 histone chaperone involved in histone dynamics during replication, transcription, and DNA repair. Overexpressed in proliferating tissues including many tumors, ASF1 has emerged as a promising therapeutic target. Here, we combine structural, computational, and biochemical approaches to design peptides that inhibit the ASF1-histone interaction. Starting from the structure of the human ASF1-histone complex, we developed a rational design strategy combining epitope tethering and optimization of interface contacts to identify a potent peptide inhibitor with a dissociation constant of 3 nM. When introduced into cultured cells, the inhibitors impair cell proliferation, perturb cell-cycle progression, and reduce cell migration and invasion in a manner commensurate with their affinity for ASF1. Finally, we find that direct injection of the most potent ASF1 peptide inhibitor in mouse allografts reduces tumor growth. Our results open new avenues to use ASF1 inhibitors as promising leads for cancer therapy.La fonction anti-silencing 1 (ASF1) est un chaperon d'histone H3-H4 conservé, impliqué dans la dynamique des histones pendant la réplication, la transcription et la réparation de l'ADN. Surexprimée dans les tissus en prolifération, y compris dans de nombreuses tumeurs, l'ASF1 est devenue une cible thérapeutique prometteuse. Ici, nous combinons des approches structurelles, informatiques et biochimiques pour concevoir des peptides qui inhibent l'interaction ASF1-histone. En partant de la structure du complexe ASF1-histone humain, nous avons mis au point une stratégie de conception rationnelle combinant la fixation des épitopes et l'optimisation des contacts d'interface pour identifier un puissant inhibiteur peptidique avec une constante de dissociation de 3 nM. Lorsqu'ils sont introduits dans des cellules en culture, les inhibiteurs entravent la prolifération cellulaire, perturbent la progression du cycle cellulaire et réduisent la migration et l'invasion des cellules d'une manière proportionnelle à leur affinité pour l'ASF1. Enfin, nous constatons que l'injection directe du plus puissant inhibiteur du peptide ASF1 dans les allogreffes de souris réduit la croissance des tumeurs. Nos résultats ouvrent de nouvelles voies pour utiliser les inhibiteurs de l'ASF1 comme des pistes prometteuses pour le traitement du cancer

TCTP contains a BH3-like domain, which instead of inhibiting, activates Bcl-xL

International audienceTranslationally Controlled Tumor Protein (TCTP) is anti-apoptotic, key in development and cancer, however without the typical Bcl2 family members' structure. Here we report that TCTP contains a BH3-like domain and forms heterocomplexes with Bcl-xL. The crystal structure of a Bcl-xL deletion variant-TCTP11-31 complex reveals that TCTP refolds in a helical conformation upon binding the BH3-groove of Bcl-xL, although lacking the h1-subregion interaction. Experiments using in vitro-vivo reconstituted systems and TCTP(+/-) mice indicate that TCTP activates the anti-apoptotic function of Bcl-xL, in contrast to all other BH3-proteins. Replacing the non-conserved h1 of TCTP by that of Bax drastically increases the affinity of this hybrid for Bcl-xL, modifying its biological properties. This work reveals a novel class of BH3-proteins potentiating the anti-apoptotic function of Bcl-xL