CMG2; anthrax toxin; Endoplasmic reticulum; toxins; TEM8; endocytosis; ubiquination; Wnt signaling; Capillary morphogenesis gene 2; tumor endothelial marker 8; systemic hyalinosis
Szczesny P, Iacovache I, Muszewska A, Ginalski K, van der Goot FG, Grynberg M (2011), Extending the aerolysin family: From bacteria to vertebrates, in PLoS ONE
, 6(6), 111-123.
Opota O, Vallet-Gely I, Vincentelli R, Kellenberger C, Iacovache I, Gonzalez MR, Roussel A, van der Goot FG, Lemaitre B (2011), Monalysin, a Novel beta-Pore-Forming Toxin from the Drosophila Pathogen Pseudomonas entomophila, Contributes to Host Intestinal Damage and Lethality, in PLOS PATHOGENS
, 7(9), 9898-1212.
Gonzalez MR, Bischofberger M, Frêche B, Ho S, Parton RG, Van der Goot FG (2011), Pore-forming toxins induce multiple cellular responses promoting survival, in Cellular Microbiology
, 13(7), 1026-1043.
Iacovache I, Degiacomi MT, Pernot L, Ho S, Schiltz M, Dal Peraro M, van der Goot FG (2011), Dual chaperone role of the c-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin, in PLoS Pathogens
, 7(7), 48-62.
Deuquet J, Lausch E, Guex N, Abrami L, Salvi S, Lakkaraju A, Ramirez MCM, Martignetti JA, Rokicki D, Bonafe L, Superti-Furga A, van der Goot FG (2011), Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors, in EMBO MOLECULAR MEDICINE
, 3(4), 208-221.
Iacovache I, Biasini M, Kowal J, Kukulski W, Chami M, van der Goot FG, Engel A, Rémigy H-W (2010), The 2DX robot: A membrane protein 2D crystallization Swiss Army knife, in Journal of Structural Biology
, 169(3), 370-378.
Iacovache I, Bischofberger M, van der Goot FG (2010), Structure and assembly of pore-forming proteins, in CURRENT OPINION IN STRUCTURAL BIOLOGY
, 20(2), 241-246.
Deuquet J, Abrami L, Difeo A, Ramirez MCM, Martignetti JA, van der Goot FG (2009), Systemic Hyalinosis Mutations in the CMG2 Ectodomain Leading to Loss of Function Through Retention in the Endoplasmic Reticulum, in HUMAN MUTATION
, 30(4), 583-589.
Abrami L, Kunz B, Iacovache I, Van Der Goot FG (2008), Palmitoylation and ubiquitination regulate exit of the Wnt signaling protein LRP6 from the endoplasmic reticulum, in Proceedings of the National Academy of Sciences of the United States of America
, 105(14), 5384-5389.
Iacovache I, van der Goot FG, Pernot L (2008), Pore formation: An ancient yet complex form of attack, in Biochimica et Biophysica Acta - Biomembranes
, 1778(7-8), 1611-1623.
Roduit C, van der Goot FG, Los Rios P, Yersin A, Steiner P, Dietler G, Catsicas S, Lafont F, Kasas S (2008), Elastic membrane heterogeneity of living cells revealed by stiff nanoscale membrane domains, in BIOPHYSICAL JOURNAL
, 94(4), 1521-1532.
BackgroundA major focus of our work has been the understanding of the structure of bacterial toxins, how they interact with mammalian cells and how the target cell respond to the toxic insults. We have concentrated on pore-forming toxins, the largest class of bacterial protein toxins (work not described in this proposal) and on the anthrax toxin, which belongs to the class of AB toxins. The B subunit is involved in binding to the target cell and escorting the A subunit to the cytoplasm. The subunit bares the enzymatic toxic activity. Anthrax toxin has two A subunits, lethal toxin -a metalloprotease that cleaves MAP kinase kinases- and edema factor -a calmodulin dependent adenylate cyclase. Toxicity is strictly dependent upon the delivery of the enzymatic subunits to the cytoplasm and thus much of our attention has been focused on understanding the molecular mechanism that mediate toxin uptake and cytoplasmic delivery. These events are largely dependent on the anthrax toxin receptors (ATRs), of which there are two: capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8). Very little is known about the structure and physiological function of these two proteins. Interestingly, mutations in CMG2 are associated with a severe, but rare, autosomal recessive human disease called Systemic Hyalinosis. The work proposed here will focus on one hand on the mode of action of the anthrax toxin and the cellular responses towards this intoxication and on the other of the structure, cellular behavior and physiological functions of anthrax toxin receptors, in particular of CMG2 and its implication in Systemic Hyalinosis. We expect extensive cross-talk and cross-fertilization between these two major subprojects.Specific aims1) Mechanism of the anthrax toxin entry and cytoplasmic delivery: we will pursue our efforts in understanding how anthrax toxin orchestrates its own uptake into cells in particular by triggering the post-translational modifications of the receptors and interacting partners. We will in particular search for novel partners and characterize the underlying interactions.2) Structure and folding of anthrax receptors (ATRs) and their interacting protein LRP6, involved in Wnt signaling: the extracellular and cytoplasmic domains of ATRs and the cytoplasmic domain of LRP6 will be produced recombinantly and their structure analyzed using various spectroscopic techniques and X-ray crystallography. In vivo folding and assembly of these proteins in the ER will be studied. Using both a proteomics and a candidate-based approach, we will in particular unravel the molecular components mediating the novel ubiquitin dependent ER quality control system that we have recently identified.3) Role and behavior of ATRs at the cellular level: since ATRs were shown to bind extracellular matrix (ECM) proteins, we will investigate their possible role in cell adhesion and spreading as well as ECM turnover. In the search of the exact role, we will search for partner proteins using a variety of techniques and perform gene-profiling experiments after expression or knock down of ATRs in cell lines and primary human fibroblasts. We will in particular pursue our studies on the interaction of ATRs with LRP6, and investigate whether a crosstalk exists between ATR mediated interaction with the ECM and Wnt signaling. 4) Study of CMG2 in model organisms: we are in the process of generating conditional knock out mice for CMG2. We will subsequently analysis the consequences of CMG2 knock down in the whole animal and in specific tissues during development of the animal. We have also launched studies on the role of ATRs in zebrafish, which express 6 ATRs related genes. We will analyze the roles of these genes in development of the embryo and determine the pathways in which they are involved.5) Molecular mechanisms of Systemic Hyalinosis: we will reproduce the various reported Systemic Hyalinosis mutations and analyze their effects of protein localization, function and turnover using tissue culture cells and if possible recomplementation in zebrafish. We are also generating two mutant mice harboring Systemic Hyalinosis point mutations in CMG2 and will investigate whether these mice will constitute an adequate animal model of the disease.Experimental designThe above described points will be tackled using a multidisciplinary approach combining biochemical (protein purification, immunoprecipitation, subcellular fractionation, proteomics,…), structural (spectroscopic techniques, crystallography,…), molecular biology (site directed mutagenesis, gene profiling, gene silencing, …), animal models (mice and fish) and morphological techniques (light and electron microscopy). We have generated the necessary strong worldwide network of collaborations and will benefits from the state-of-the-art facilities available in the Lemanic region (mouse facilities, microscopy, proteomics, genomics,…).Expected value of the proposed projectThe proposed work will provide a better understanding of the mode of action of one of the main virulence factor involved in anthrax and on the uptake of bacterial toxins in general. Moreover, the work will provide novel information on the role of ATRs, their interaction with LRP6 and Wnt signaling pathways, and the mechanism underlying Systemic Hyalinosis, all of which are almost virgin areas of research.