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Accueil > Départements > Biochimie, Biophysique et Biologie Structurale > Christophe LE CLAINCHE & Louis RENAULT : Dynamique du cytosquelette et motilité

Functional and structural studies of bacterial virulence factors activated by actin, or of multimodular proteins with intrinsically disordered actin-binding domains

Louis RENAULT

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Functional and structural studies of enzymes involved in intracellular host-pathogen interactions, innate immunity regulation or antibiotic resistance, or of multimodular protein architectures with intrinsically disordered actin-binding domains

Contact


RENAULT Louis [Chargé de Recherche - CNRS]
Equipe Le Clainche C./Renault L. - Dynamique du cytosquelette et motilité [Responsable]
01 69 82 34 89 Gif - Bât 34

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Main research projects

- Biochemical, structural and functional studies of enzymes involved in intracellular host-pathogen interactions, innate immunity regulation or antibiotic resistance.

We are also analyzing the structural basis for the activities and specificities of enzymes that are involved in intracellular host-pathogen interactions, innate immunity regulation, such as virulence factors of opportunistic bacterial pathogens that are targeting or activated by actin in infected eukaryotic cells, or in antibiotic resistance, such as bacterial rRNA-methyltransferases that promote bacterial resistance against mycinosylated macrolide antibiotics.

Example of ongoing project with recent results :
Biochemical and structural study of the functional specificities of a new family of bacterial nucleotidyl cyclase toxins activated by actin in host cells.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes severe nosocomial infections in immune-compromised patients or chronic infections in people with cystic fibrosis. Its exoenzyme Y (ExoY) is one of its key virulence factors injected into host target cells [Hauser, A. R. (2009) Nat Rev Microbiol 7, 654-65]. In addition, ExoY-related (ExoY-like) toxins have been recently identified in the genomes of various gram-negative bacteria that represent emerging human or animal pathogens, and where the nucleotidyl cyclase toxins may be essential for the bacterial virulence [Ziolo, K. J., et al. (2014) Infect Immun 82, 2148-57]. ExoY and ExoY-like toxins define a new and atypical subfamily of bacterial nucleotidyl cyclase toxins that is poorly understood.

Inactive in bacteria, these toxins are specifically activated after their secretion inside host cells by interacting with a eukaryotic cofactor, markor of their host cells. Since the discovery of ExoY in 1998, ignorance of its activation mechanism has hindered studies of its activity. In collaboration with the team of Undine Mechold & Daniel Ladant from Pasteur Institute in Paris, France, we recently identified actin as being the common eukaryotic cofactor required for activation of several ExoY/ExoY-like toxins in host cells [Belyy A. et al. (2016) Nature Communications 2016 Dec 5 ; 7:13582]. Pseudomonas aeruginosa ExoY thus represents the first known bacterial virulence factor to be activated specifically with actin filaments. P. aeruginosa ExoY association along actin filaments promotes its guanlylate and adenylate cyclase activity by more than 10,000 fold and alters the turnover of actin filaments in vitro and in transfected eukaryotic cells [Belyy A. et al. (2016)].
In collaboration with teams at Pasteur Institute, Paris, we aim to characterize at the molecular level the precise role and functional specificities of this novel class of toxins in bacterial infections by P. aeruginosa and various pathogenic gram-negative organisms, with the long-term goal to explore their potential as novel anti-infective agents.

Keywords : host-pathogen interactions, bacteriology, virulence factors, bacterial RNA-binding enzymes, innate immunity regulation, antibiotic resistance.

- Biochemical and structural studies of the multifunctionality of multidomain actin binding proteins with intrinsically disordered domains.

In eukaryotic cells, a tight control of actin assembly dynamics by actin binding proteins (ABPs) is central to many cytoskeletal processes such as muscle contraction, cell adhesion and motility and intracellular trafficking. More recently, actin appears to be directly involved in nuclear processes such as chromatin remodeling and gene transcription. ABPs often display complex multi-modular protein architectures that support their multiple interactions and functions with many different partners. Here we study how multi-domain ABPs use multifunctional intrinsically disordered regions (IDRs) to regulate the dynamics of actin filament assembly and disassembly.

Intrinsically disordered proteins/domains define a new class of functional proteins/domains whose structural, interfacial and regulatory properties remain to be determined in many cellular processes. They are more abundant in eukaryotic proteins but remain however difficult to identify and characterize in vitro [Tompa P. (2012) Trends Biochem Sci 37(12):509-16]. Indeed, they do not adopt a unique and stable tertiary structure in solution, but display versatile adaptability in binding, often weak but specific binding, and frequent regulation by post-translational modifications or alternative splicings. The interest to understand acutely their molecular mechanisms at protein-protein interfaces is further accentuated by the involvement of numerous IDPs in many human diseases, such as amyloidoses and neurodegenerative diseases, but also cancer, cardiovascular disease, and diabetes [Uversky VN (2010) Expert Rev. Proteomics 7(4):543-64 ; Tompa P. (2012) Trends Biochem Sci 37(12):509-16].

We aim at understanding in detail the structure-function relationship and molecular mechanisms of regulation specific to multi-modular organizations that exploit intrinsically disordered domains, as small, ubiquitous actin-binding WH2 domains, to interact with both actin monomers (called G-actin) and / or filament actin (F-actin) subunits. Our general objective is to understand at the atomic scale the coordinated interactions of IDR-containing ABPs, to reconstitute appropriate integrated structural models and systems in vitro that approach the cellular context and reveal the cross-talks, synergies or antagonisms in actin assembly dynamics between different domains of ABPs and/or key cytoskeletal or nucleoskeletal proteins.

Keywords : intrinsically disordered proteins, WH2 (WASP Homology 2) domains, multifunctional actin binding proteins, Formin, nucleoskeleton.

Main approaches

- Bioinformatic and structural analysis
- Expression and purification of recombinant proteins, protein labeling
- Biochemical analysis (kinetics of actin polymerization, functional assays in actin self-assembly, affinity measurements, hydrodynamic characterizations and modeling ...)
- Crystallization, structural determination and analysis by crystallography and small angle X-ray scattering (SAXS) in solution, combined with complementary structural analyses like NMR in collaborations

WH2 Approaches

FIGURE 2 : Example of questions that we address and approaches we develop in the lab to decipher the structural bases for the multifunctionality of multi-modular proteins containing small, intrinsically-disordered actin binding WH2 domains/repeats, and involved in cell migration, morphogenesis, intracellular trafficking or pathogen infections.

Master internships/ Open positions

The research projects are opened each year to students looking for M1/M2 internships with strong interests in understanding in atomic detail the structure-function relationships of complex protein architectures, their dynamics and potentially their inhibition in pathological contexts by combining extensively biochemical and structural approaches.

Ongoing collaborations

- Undine Mechold, Institut Pasteur, CNRS URA3528, Unité de Biochimie des Interactions macromoléculaires, Département de Biologie Structurale et Chimie, Paris, France.
- Carine van Heijenoort, Laboratoire de Chimie et Biologie Structurales, Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France.
- Sophie Zinn-Justin, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, CEA Saclay, Gif-sur-Yvette, France.
- Dominique Fourmy, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Gif-sur-Yvette, France.
- Metello Innocenti, The Netherlands Cancer Institute (NKI), Amsterdam, The Netherlands.

Publications since 2014

FEBS Lett. 2016 Oct;590(20):3690-3699
Mutations in actin used for structural studies partially disrupt β-thymosin/WH2 domains interaction. Authors: Deville C, Girard-Blanc C, Assrir N, Nhiri N, Jacquet E, Bontems F, Renault L, Petres S, van Heijenoort C [PubMed]

Vitam Horm. 2016;102:25-54
Intrinsic, Functional, and Structural Properties of β-Thymosins and β-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling. Authors: Renault L [PubMed]

ACS Chem Biol. 2015 Dec 18;10(12):2733-42
Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Self-assembly Properties. Authors: Herrada I, Samson C, Velours C, Renault L, Östlund C, Chervy P, Puchkov D, Worman HJ, Buendia B, Zinn-Justin S [PubMed]

Nat Commun. 2016 Dec 05;7:13582
Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins. Authors: Belyy A, Raoux-Barbot D, Saveanu C, Namane A, Ogryzko V, Worpenberg L, David V, Henriot V, Fellous S, Merrifield C, Assayag E, Ladant D, Renault L, Mechold U [PubMed]

Selected publications prior to 2014

Between 2013-2010

• Dias J., Renault L.#, Pérez J., Mirande M.# (2013) Small-angle X-ray solution scattering study of the multi-aminoacyl-tRNA synthetase complex reveals an elongated and multi-armed particle.
J. Biol. Chem. 288(33):23979-89. (# : corresponding authors)

• Renault, L#, Deville, C., van Heijenoort C. (2013) Structural features and interfacial properties of WH2, β-Thymosin domains and other intrinsically disordered domains in the regulation of actin cytoskeleton dynamics.
Cytoskeleton (Hoboken) 70(11):686-705. (# : corresponding author)

• D. Didry, F.X. Cantrelle, C. Husson, P. Roblin, A. M. Eswara Moorthy, J. Perez, C. Le Clainche, M. Hertzog, E. Guittet, M.F. Carlier, C. van Heijenoort# and L. Renault#. (2012) How a Single Residue in Individual ß-Thymosin/WH2 Domains Controls their Functions in Actin Assembly.
EMBO J. 31(4):1000-13 (# : corresponding authors)

• C. Husson, L. Renault, D. Didry, D. Pantaloni, M.F. Carlier#. (2011) Cordon-Bleu uses WH2 domains as multifunctional dynamizers of actin filament assembly.
Molecular Cell 43, 464-77.

• C. Husson, F.X. Cantrelle, P. Roblin, D. Didry, K.H. Le, J. Perez, E. Guittet, C. Van Heijenoort#, L. Renault# and M.F. Carlier# (2010) Multifunctionality of the ß-Thymosin/WH2 module : G-actin sequestration, actin filament growth, nucleation and severing.
Annals of the New York Academy of Sciences 1194, 44-52. review. (# : corresponding authors).

Between 2009-1998

Multifunctionality of small intrinsically disordered actin-binding WH2 repeats

• Bosch M, Le KH, Bugyi B, Correia JJ, Renault L#, Carlier MF#. (2007) Analysis of the Function of Spire in Actin Assembly and Its Synergy with Formin and Profilin.
Molecular Cell 28(4), 555-568. (# : corresponding authors).

Interferon-gamma inducible GTP-binding proteins involved in innate immunity regulation, or proteins / virulence factors of pathogens

• Ghosh A., Praefcke G. J. K., Renault L.#, Wittinghofer A.#, Herrmann C. (2006) How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP.
Nature 440, 101-4. (# : corresponding authors).

• Hible G., Christova P., Renault L., Seclaman E., Thompson A., Girard E., Munier-Lehmann H., and Cherfils J. (2006) Unique GMP-binding site in Mycobacterium tuberculosis guanosine monophosphate kinase.
Proteins 62, 489-500.

• Würtele M., Renault L., Barbieri J.T., Wittinghofer A. & Wolf E. (2001) Structure of the ExoS GTPase activating domain.
FEBS L. 491, 26-29.

• Prakash, B., Praefcke, G.J.K., Renault, L., Wittinghofer, A. & Hermann, C. (2000) Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins.
Nature 403, 567-71.

Eukaryotic factors activating GTP-binding proteins involved in intracellular trafficking and their inhibition mechanisms by mutations or a small fungal metabolite

• Renault L., Guibert B., Cherfils J. (2003) Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor.
Nature 426, 525-530.

• Renault L., Kuhlmann J., Henkel A. & Wittinghofer A. (2001) Structural basis for guanine nucleotide exchange on Ran by the Regulator of Chromosome Condensation (RCC1).
Cell 105, 245-255.

• Renault, L., Nassar, N., Vetter, I., Becker, J., Klebe, C., Roth, M. & Wittinghofer, A. (1998) The 1.7 Å crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller.
Nature 392, 97-101.

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