Publications by authors named "Béatrice Kunz"

13 Publications

  • Page 1 of 1

Ligand Binding to the Collagen VI Receptor Triggers a Talin-to-RhoA Switch that Regulates Receptor Endocytosis.

Dev Cell 2020 05;53(4):418-430.e4

Faculty of Life Sciences, Global Health Institute, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland. Electronic address:

Capillary morphogenesis gene 2 (CMG2/ANTXR2) is a cell surface receptor for both collagen VI and anthrax toxin. Biallelic loss-of-function mutations in CMG2 lead to a severe condition, hyaline fibromatosis syndrome (HFS). We have here dissected a network of dynamic interactions between CMG2 and various actin interactors and regulators, describing a different behavior from other extracellular matrix receptors. CMG2 binds talin, and thereby the actin cytoskeleton, only in its ligand-free state. Extracellular ligand binding leads to src-dependent talin release and recruitment of the actin cytoskeleton regulator RhoA and its effectors. These sequential interactions of CMG2 are necessary for the control of oriented cell division during fish development. Finally, we demonstrate that effective switching between talin and RhoA binding is required for the intracellular degradation of collagen VI in human fibroblasts, which explains why HFS mutations in the cytoskeleton-binding domain lead to dysregulation of extracellular matrix homeostasis.
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http://dx.doi.org/10.1016/j.devcel.2020.04.015DOI Listing
May 2020

Identification and dynamics of the human ZDHHC16-ZDHHC6 palmitoylation cascade.

Elife 2017 08 15;6. Epub 2017 Aug 15.

Global Health Institute, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

S-Palmitoylation is the only reversible post-translational lipid modification. Knowledge about the DHHC palmitoyltransferase family is still limited. Here we show that human ZDHHC6, which modifies key proteins of the endoplasmic reticulum, is controlled by an upstream palmitoyltransferase, ZDHHC16, revealing the first palmitoylation cascade. The combination of site specific mutagenesis of the three ZDHHC6 palmitoylation sites, experimental determination of kinetic parameters and data-driven mathematical modelling allowed us to obtain detailed information on the eight differentially palmitoylated ZDHHC6 species. We found that species rapidly interconvert through the action of ZDHHC16 and the Acyl Protein Thioesterase APT2, that each species varies in terms of turnover rate and activity, altogether allowing the cell to robustly tune its ZDHHC6 activity.
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http://dx.doi.org/10.7554/eLife.27826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582869PMC
August 2017

CMG2/ANTXR2 regulates extracellular collagen VI which accumulates in hyaline fibromatosis syndrome.

Nat Commun 2017 06 12;8:15861. Epub 2017 Jun 12.

Global Health Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne 1015, Switzerland.

Loss-of-function mutations in capillary morphogenesis gene 2 (CMG2/ANTXR2), a transmembrane surface protein, cause hyaline fibromatosis syndrome (HFS), a severe genetic disorder that is characterized by large subcutaneous nodules, gingival hypertrophy and severe painful joint contracture. Here we show that CMG2 is an important regulator of collagen VI homoeostasis. CMG2 loss of function promotes accumulation of collagen VI in patients, leading in particular to nodule formation. Similarly, collagen VI accumulates massively in uteri of Antxr2 mice, which do not display changes in collagen gene expression, and leads to progressive fibrosis and sterility. Crossing Antxr2 with Col6a1 mice leads to restoration of uterine structure and reversion of female infertility. We also demonstrate that CMG2 may act as a signalling receptor for collagen VI and mediates its intracellular degradation.
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http://dx.doi.org/10.1038/ncomms15861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472780PMC
June 2017

Ubiquitin-dependent folding of the Wnt signaling coreceptor LRP6.

Elife 2016 10 18;5. Epub 2016 Oct 18.

Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

Many membrane proteins fold inefficiently and require the help of enzymes and chaperones. Here we reveal a novel folding assistance system that operates on membrane proteins from the cytosolic side of the endoplasmic reticulum (ER). We show that folding of the Wnt signaling coreceptor LRP6 is promoted by ubiquitination of a specific lysine, retaining it in the ER while avoiding degradation. Subsequent ER exit requires removal of ubiquitin from this lysine by the deubiquitinating enzyme USP19. This ubiquitination-deubiquitination is conceptually reminiscent of the glucosylation-deglucosylation occurring in the ER lumen during the calnexin/calreticulin folding cycle. To avoid infinite futile cycles, folded LRP6 molecules undergo palmitoylation and ER export, while unsuccessfully folded proteins are, with time, polyubiquitinated on other lysines and targeted to degradation. This ubiquitin-dependent folding system also controls the proteostasis of other membrane proteins as CFTR and anthrax toxin receptor 2, two poor folders involved in severe human diseases.
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http://dx.doi.org/10.7554/eLife.19083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102578PMC
October 2016

Palmitoylated calnexin is a key component of the ribosome-translocon complex.

EMBO J 2012 Apr 7;31(7):1823-35. Epub 2012 Feb 7.

Global Health Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

A third of the human genome encodes N-glycosylated proteins. These are co-translationally translocated into the lumen/membrane of the endoplasmic reticulum (ER) where they fold and assemble before they are transported to their final destination. Here, we show that calnexin, a major ER chaperone involved in glycoprotein folding is palmitoylated and that this modification is mediated by the ER palmitoyltransferase DHHC6. This modification leads to the preferential localization of calnexin to the perinuclear rough ER, at the expense of ER tubules. Moreover, palmitoylation mediates the association of calnexin with the ribosome-translocon complex (RTC) leading to the formation of a supercomplex that recruits the actin cytoskeleton, leading to further stabilization of the assembly. When formation of the calnexin-RTC supercomplex was affected by DHHC6 silencing, mutation of calnexin palmitoylation sites or actin depolymerization, folding of glycoproteins was impaired. Our findings thus show that calnexin is a stable component of the RTC in a manner that is exquisitely dependent on its palmitoylation status. This association is essential for the chaperone to capture its client proteins as they emerge from the translocon, acquire their N-linked glycans and initiate folding.
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http://dx.doi.org/10.1038/emboj.2012.15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321195PMC
April 2012

Endocytosis of the anthrax toxin is mediated by clathrin, actin and unconventional adaptors.

PLoS Pathog 2010 Mar 5;6(3):e1000792. Epub 2010 Mar 5.

Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Faculty of Life Sciences, Lausanne, Switzerland.

The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a beta-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin.
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http://dx.doi.org/10.1371/journal.ppat.1000792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832758PMC
March 2010

Anthrax toxin triggers the activation of src-like kinases to mediate its own uptake.

Proc Natl Acad Sci U S A 2010 Jan 4;107(4):1420-4. Epub 2010 Jan 4.

Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.

AB-type toxins, like other bacterial toxins, are notably opportunistic molecules. They rely on target cell receptors to reach the appropriate location within the target cell where translocation of their enzymatic subunits occurs. The anthrax toxin, however, times its own uptake, suggesting that toxin binding triggers specific signaling events. Here we show that the anthrax toxin triggers tyrosine phosphorylation of its own receptors, capillary morphogenesis gene 2 and tumor endothelial marker 8, which are not endowed with intrinsic kinase activity. This is required for efficient toxin uptake because endocytosis of the mutant receptor lacking the cytoplasmic tyrosine residues is strongly delayed. Phosphorylation of the receptors was dependent on src-like kinases, which where activated upon toxin binding. Importantly, src-dependent phosphorylation of the receptor was required for its subsequent ubiquitination, which in turn was required for clathrin-mediated endocytosis. Consistently, we found that uptake of the anthrax toxin and processing of the lethal factor substrate MEK1 are inhibited by silencing of src and fyn, as well as in src and fyn knockout cells.
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http://dx.doi.org/10.1073/pnas.0910782107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824395PMC
January 2010

Functional interactions between anthrax toxin receptors and the WNT signalling protein LRP6.

Cell Microbiol 2008 Dec 20;10(12):2509-19. Epub 2008 Aug 20.

Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Station 15, CH 1015 Lausanne, Switzerland.

To exert its activity, anthrax toxin must be endocytosed and its enzymatic toxic subunits delivered to the cytoplasm. It has been proposed that, in addition to the anthrax toxin receptors (ATRs), lipoprotein-receptor-related protein 6 (LRP6), known for its role in Wnt signalling, is also required for toxin endocytosis. These findings have however been challenged. We show that LRP6 can indeed form a complex with ATRs, and that this interaction plays a role both in Wnt signalling and in anthrax toxin endocytosis. We found that ATRs control the levels of LRP6 in cells, and thus the Wnt signalling capacity. RNAi against ATRs indeed led to a drastic decrease in LRP6 levels and a subsequent drop in Wnt signalling. Conversely, LRP6 plays a role in anthrax toxin endocytosis, but is not essential. We indeed found that toxin binding triggered tyrosine phosphorylation of LRP6, induced its redistribution into detergent-resistant domains, and its subsequent endocytosis. RNAis against LRP6 strongly delayed toxin endocytosis. As the physiological role of ATRs is probably to interact with the extracellular matrix, our findings raise the interesting possibility that, through the ATR-LRP6 interaction, adhesion to the extracellular matrix could locally control Wnt signalling.
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http://dx.doi.org/10.1111/j.1462-5822.2008.01226.xDOI Listing
December 2008

Palmitoylation and ubiquitination regulate exit of the Wnt signaling protein LRP6 from the endoplasmic reticulum.

Proc Natl Acad Sci U S A 2008 Apr 31;105(14):5384-9. Epub 2008 Mar 31.

Global Health Institute, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Station 15, CH-1015 Lausanne, Switzerland.

Canonical Wnt signaling is initiated by binding of Wnt proteins to members of the Frizzled family and subsequent complex formation with lipoprotein receptor-related proteins 5/6 (LRP5/6). Here, we show that LRP6 is palmitoylated on a juxtamembranous cysteine and that palmitoylation is required for exit from the endoplasmic reticulum (ER). We propose that palmitoylation serves to tilt the long, 23-residue transmembrane domain of LRP6 with respect to the plane of membrane to prevent a hydrophobic mismatch and subsequent recognition by the ER quality control. In support of this model, a palmitoylation-deficient LRP6 mutant could be rescued from ER retention by deletion of two to four residues in the transmembrane domain. Importantly, we found that palmitoylation-deficient LRP6 was retained in the ER by a completely novel monoubiquitination-dependent ER retention mechanism. Mutation of a specific lysine indeed abolished ubiquitination of palmitoylation-deficient LRP6 and led to a rescue from ER retention. Finally, at the cell surface, we found that interplay between palmitoylation and ubiquitination was necessary for efficient Wnt signaling.
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http://dx.doi.org/10.1073/pnas.0710389105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291096PMC
April 2008

Simple and efficient isolation of hematopoietic stem cells from H2K-zFP transgenic mice.

Stem Cells 2005 Nov-Dec;23(10):1617-25. Epub 2005 Aug 9.

Department of Life Science, Swiss Federal Institute of Technology, Lausanne, Switzerland.

We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFP(bright) cells are also c-kit(high), Sca-1(high), Lin(neg), Flk-2(neg), which is a bona fide phenotype for long-term HSCs. Double-sorted zFP(bright) HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages.
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http://dx.doi.org/10.1634/stemcells.2004-0374DOI Listing
March 2006

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells.

Chembiochem 2005 Jul;6(7):1263-9

Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering, 1015 Lausanne, Switzerland.

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.
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http://dx.doi.org/10.1002/cbic.200400431DOI Listing
July 2005

Initiation and limitation of Ly-49A NK cell receptor acquisition by T cell factor-1.

J Immunol 2003 Jul;171(2):769-75

Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland.

The establishment of clonally variable expression of MHC class I-specific receptors by NK cells is not well understood. The Ly-49A receptor is used by approximately 20% of NK cells, whereby most cells express either the maternal or paternal allele and few express simultaneously both alleles. We have previously shown that NK cells expressing Ly-49A were reduced or almost absent in mice harboring a single or no functional allele of the transcription factor T cell factor-1 (TCF-1), respectively. In this study, we show that enforced expression of TCF-1 in transgenic mice yields an expanded Ly-49A subset. Even though the frequencies of Ly-49A(+) NK cells varied as a function of the TCF-1 dosage, the relative abundance of mono- and biallelic Ly-49A cells was maintained. Mono- and biallelic Ly-49A NK cells were also observed in mice expressing exclusively a transgenic TCF-1, i.e., expressing a fixed amount of TCF-1 in all NK cells. These findings suggest that Ly-49A acquisition is a stochastic event due to limiting TCF-1 availability, rather than the consequence of clonally variable expression of the endogenous TCF-1 locus. Efficient Ly-49A acquisition depended on the expression of a TCF-1 isoform, which included a domain known to associate with the TCF-1 coactivator beta-catenin. Indeed, the proximal Ly-49A promoter was beta-catenin responsive in reporter gene assays. We thus propose that Ly-49A receptor expression is induced from a single allele in occasional NK cells due to a limitation in the amount of a transcription factor complex requiring TCF-1.
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http://dx.doi.org/10.4049/jimmunol.171.2.769DOI Listing
July 2003

A role for the src family kinase Fyn in NK cell activation and the formation of the repertoire of Ly49 receptors.

Eur J Immunol 2002 03;32(3):773-82

Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland.

NK cell function is regulated by a dual receptor system, which integrates signals from triggering receptors and MHC class I-specific inhibitory receptors. We show here that the src family kinase Fyn is required for efficient, NK cell-mediated lysis of target cells, which lack both self-MHC class I molecules and ligands for NKG2D, an activating NK cell receptor. In contrast, NK cell inhibition by the MHC class I-specific receptor Ly49A was independent of Fyn, suggesting that Fyn is specifically required for NK cell activation via non-MHC receptor(s). Compared to wild type, significantly fewer Fyn-deficient NK cells expressed the inhibitory Ly49A receptor. The presence of a transgenic Ly49A receptor together with its H-2(d) ligand strongly reduced the usage of endogenous Ly49 receptors in Fyn-deficient mice. These data suggest a model in which the repertoire of inhibitory Ly49 receptors is formed under the influenced of Fyn-dependent NK cell activation as well as the respective MHC class I environment. NK cells may acquire Ly49 receptors until they generate sufficient inhibitory signals to balance their activation levels. Such a process would ensure the induction of NK cell self-tolerance.
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http://dx.doi.org/10.1002/1521-4141(200203)32:3<773::AID-IMMU773>3.0.CO;2-UDOI Listing
March 2002