Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors

Hyaline Fibromatosis Syndrome (HIS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized lg-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the lg-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Molecular Mechanisms Underlying Hyaline Fibromatosis Syndrome

Julie Deuquet Ariosa

Hyaline Fibromatosis Syndrome (HFS) is a rare inherited disease that is characterized by an accumulation of an unidentified hyaline material, largely affecting connective tissues. Patients afflicted with HFS present a wide range of clinical symptoms such as nodules, papules, hyperplasia of the gingiva, joint contractions, thickness and hyperpigmentation of the skin, and osteopenia. Depending of the severity and the delay of appearance of these symptoms, the life expectancy varies between 2 years and > 30 years. Death earlier in life is due to recurrent diarrhea and chest infections of unknown origin. In 2003, the gene responsible for HFS was identified to be Capillary Morphoqenesis Gene 2 (CMG2), a gene upregulated during capillary morphogenesis in three-dimensional collagen matrices. This gene encodes a type I transmembrane glycoprotein composed of a von Willebrand (vWA) domain followed by an immunoglobulin (Ig)-like domain in its extracellular part, a single α-helix spanning transmembrane domain, and a 148 amino acids cytoplasmic tail predicted as unstructured. Even though the physiological function of CMG2 remains to be elucidated, it has been shown that this protein induces endothelial proliferation, and dictates cell polarization during embryogenesis in a zebrafish model. Besides its physiological function, CMG2 was found to be an anthrax toxin receptor, thus its alternative name ANTXR2, and has been extensively studied in this context. Genetic analysis has led to the identification of 34 CMG2 mutations in HFS patients. The molecular mechanisms underlying the disease have however not yet been identified. During the five years of my thesis, we were interested in understanding the cellular consequences of these mutations and in getting a better understanding of the physiological function of CMG2. I first showed that most of the HFS mutations in the vWA domain lead to alterations in folding of the domain, either in terms of kinetics or of structure, leading to recognition of the protein by the ER quality control (ERQC) and subsequent degradation by the ERAD pathway. A direct consequence of this retention is the loss of function at the cell surface or other cellular sites. In a second study, I studied the effect of novel HFS mutations that our collaborators and we identified in the CMG2 Ig-like domain. Since the structure of this domain was uncharacterized, we first generated a model of the domain and found that it has an Ig-like fold, despite the very low sequence homology, and harbors two disulfide bonds essential for folding and stabilization of the domain. HFS mutations localized to this domain led to aberrant intermolecular disulfide binding, ER retention and enhanced ERAD when compared to the vWA domain HFS mutations as shown in patients fibroblasts. The consequential loss of CMG2 could be partly alleviated by treating cells with proteasome inhibitor, such as Bortezomib, illustrating that ERAD components are potential therapeutic targets for HFS. As most of the HFS mutations in the ectodomain of CMG2 trigger this ER retention during the CMG2 biosynthesis, we then addressed this process by studying one of the most described post-translational modifications occurring in the ER for protein folding, namely the N-glycosylation. We found that the two potential CMG2 N-glycosylation sites, N250 and N260, are both occupied. Neither of the two sites is essential for ER exit. Glycosylation of CMG2 on N260 is however important since the expression levels of the N260A mutant were significant lower that for the WT protein, despite equivalent or higher synthesis, suggesting that glycosylation at this site is important for efficient folding. Finally, in order to better understand the CMG2 function, we investigated what the physiological ligands could be, assuming it is a receptor as suggested by its role in anthrax infection. We identified type VI collagen as a potential ligand in-vitro that activates CMG2 in-vivo, by phosphorylation of CMG2 cytoplasmic tail. This CMG2 activation upon type VI collagen binding could be a signal for ligand-triggered endocytosis, as is has been described with the protective antigen (PA) of anthrax toxin. The results of this work allow us to gain insights in the molecular mechanisms underlying HFS. Our studies indicate that the identification of the genotype-phenotype correlation of this disease is important for proper diagnosis and clinical approaches, and also that a therapeutic strategy based on proteasome inhibitors, such as Bortezomib, could be envisioned to treat some HFS symptoms in order to improve the life conditions of the patients.

EPFL2011

Systemic hyalinosis mutations in the CMG2 ectodomain leading to loss of function through retention in the endoplasmic reticulum

Julie Deuquet Ariosa, Laurence Gouzi Abrami, Françoise Gisou van der Goot Grunberg

Systemic hyalinosis is an autosomal recessive disease that encompasses two allelic syndromes, infantile systemic hyalinosis (ISH) and juvenile hyaline fibromatosis (JHF), which are caused by mutations in the CMG2 gene. Here we have analyzed the cellular consequences of five patient-derived point mutations in the extracellular von Willebrand domain or the transmembrane domain of the CMG2 protein. We found that four of the mutations led to retention of the protein in the endoplasmic reticulum (ER), albeit through different mechanisms. Analysis of recombinant CMG2 von Willebrand factor A (vWA) domains, to which three of the mutations map, indicated that the mutations did not prevent proper folding and ligand binding, suggesting that, in vivo, slow folding, rather than misfolding, is responsible for ER retention. Our work shows that systemic hyalinosis can be qualified as a conformational disease, at least for the mutations that have been mapped to the extracellular and transmembrane domains. The long ER half-life and the ligand binding ability of the mutated von Willebrand domains suggest that treatments based on chemical chaperones could be beneficial.

Wiley-Blackwell2009

Advanced Fluorescence Microscopy to Study Plasma Membrane Protein Dynamics

Joachim Piguet

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.