Publications by authors named "Laurence Abrami"

35 Publications

Palmitoylated acyl protein thioesterase APT2 deforms membranes to extract substrate acyl chains.

Nat Chem Biol 2021 04 11;17(4):438-447. Epub 2021 Mar 11.

Global Health Institute, School of Life Sciences, EPFL, Lausanne, Switzerland.

Many biochemical reactions require controlled recruitment of proteins to membranes. This is largely regulated by posttranslational modifications. A frequent one is S-acylation, which consists of the addition of acyl chains and can be reversed by poorly understood acyl protein thioesterases (APTs). Using a panel of computational and experimental approaches, we dissect the mode of action of the major cellular thioesterase APT2 (LYPLA2). We show that soluble APT2 is vulnerable to proteasomal degradation, from which membrane binding protects it. Interaction with membranes requires three consecutive steps: electrostatic attraction, insertion of a hydrophobic loop and S-acylation by the palmitoyltransferases ZDHHC3 or ZDHHC7. Once bound, APT2 is predicted to deform the lipid bilayer to extract the acyl chain bound to its substrate and capture it in a hydrophobic pocket to allow hydrolysis. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated deacylation of substrates throughout the endomembrane system.
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http://dx.doi.org/10.1038/s41589-021-00753-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610442PMC
April 2021

Akt Is S-Palmitoylated: A New Layer of Regulation for Akt.

Front Cell Dev Biol 2021 15;9:626404. Epub 2021 Feb 15.

Departamento de Fisiología, Biología Molecular y Celular (DFBMC), Facultad de Ciencias Exactas y Naturales (FCEN), Universidad de Buenos Aires (UBA), Buenos Aires, Argentina.

The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in Akt1 is important for proper its function, since Akt1-C344S was unable to support adipocyte cell differentiation These results add an unexpected new layer to the already complex Akt molecular code, improving our understanding of cell decision-making mechanisms such as cell survival, differentiation and death.
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http://dx.doi.org/10.3389/fcell.2021.626404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917195PMC
February 2021

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

Hemagglutinin of Influenza A, but not of Influenza B and C viruses is acylated by ZDHHC2, 8, 15 and 20.

Biochem J 2020 01;477(1):285-303

Institute of Virology, Free University Berlin, 14163 Berlin, Germany.

Hemagglutinin (HA), a glycoprotein of Influenza A viruses and its proton channel M2 are site-specifically modified with fatty acids. Whereas two cysteines in the short cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to one cysteine located at the cytoplasmic border of the transmembrane region (TMR). M2 is palmitoylated at a cysteine positioned in an amphiphilic helix near the TMR. The enzymes catalyzing acylation of HA and M2 have not been identified, but zinc finger DHHC domain-containing (ZDHHC) palmitoyltransferases are candidates. We used a siRNA library to knockdown expression of each of the 23 human ZDHHCs in HA-expressing HeLa cells. siRNAs against ZDHHC2 and 8 had the strongest effect on acylation of HA as demonstrated by Acyl-RAC and confirmed by 3H-palmitate labeling. CRISPR/Cas9 knockout of ZDHHC2 and 8 in HAP1 cells, but also of the phylogenetically related ZDHHCs 15 and 20 strongly reduced acylation of group 1 and group 2 HAs and of M2, but individual ZDHHCs exhibit slightly different substrate preferences. These ZDHHCs co-localize with HA at membranes of the exocytic pathway in a human lung cell line. ZDHHC2, 8, 15 and 20 are not required for acylation of the HA-esterase-fusion protein of Influenza C virus that contains only stearate at one transmembrane cysteine. Knockout of these ZDHHCs also did not compromise acylation of HA of Influenza B virus that contains two palmitoylated cysteines in its cytoplasmic tail. Results are discussed with respect to the acyl preferences and possible substrate recognition features of the identified ZDHHCs.
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http://dx.doi.org/10.1042/BCJ20190752DOI Listing
January 2020

Dynamic Radiolabeling of S-Palmitoylated Proteins.

Methods Mol Biol 2019 ;2009:111-127

Global Health Institute, School of Life Sciences, EPFL, Lausanne, Switzerland.

Proteins can be radiolabeled either during synthesis, typically using S-cysteine/methionine (S-Cys/Met), or after synthesis, by adding a radiolabeled posttranslational modification. Here we describe how protein S-palmitoylation, and its dynamics, can be monitored by H-palmitate labeling and how the importance of S-palmitoylation in protein biogenesis and turnover can be investigated using S-Cys/Met pulse-chase metabolic labeling. Proteins frequently have multiple palmitoylation sites. The importance thereof on the design and interpretation of metabolic labeling experiments is discussed.
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http://dx.doi.org/10.1007/978-1-4939-9532-5_9DOI Listing
March 2020

Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion.

PLoS Pathog 2018 12 10;14(12):e1007471. Epub 2018 Dec 10.

Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.

Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by 3H-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle.
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http://dx.doi.org/10.1371/journal.ppat.1007471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307819PMC
December 2018

Targeting STING with covalent small-molecule inhibitors.

Nature 2018 07 4;559(7713):269-273. Epub 2018 Jul 4.

Global Health Institute, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland.

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway. Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.
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http://dx.doi.org/10.1038/s41586-018-0287-8DOI Listing
July 2018

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

Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin.

PLoS Comput Biol 2016 Feb 22;12(2):e1004774. Epub 2016 Feb 22.

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

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non-palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6.
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http://dx.doi.org/10.1371/journal.pcbi.1004774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765739PMC
February 2016

SwissPalm: Protein Palmitoylation database.

F1000Res 2015 16;4:261. Epub 2015 Jul 16.

Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, CH-1015, Switzerland.

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation.
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http://dx.doi.org/10.12688/f1000research.6464.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4544385PMC
September 2015

Hijacking multivesicular bodies enables long-term and exosome-mediated long-distance action of anthrax toxin.

Cell Rep 2013 Nov 14;5(4):986-96. Epub 2013 Nov 14.

Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Station 19, 1015 Lausanne, Switzerland.

Anthrax lethal toxin is a classical AB toxin comprised of two components: protective antigen (PA) and lethal factor (LF). Here, we show that following assembly and endocytosis, PA forms a channel that translocates LF, not only into the cytosol, but also into the lumen of endosomal intraluminal vesicles (ILVs). These ILVs can fuse and release LF into the cytosol, where LF can proteolyze and disable host targets. We find that LF can persist in ILVs for days, fully sheltered from proteolytic degradation, both in vitro and in vivo. During this time, ILV-localized LF can be transmitted to daughter cells upon cell division. In addition, LF-containing ILVs can be delivered to the extracellular medium as exosomes. These can deliver LF to the cytosol of naive cells in a manner that is independent of the typical anthrax toxin receptor-mediated trafficking pathway, while being sheltered from neutralizing extracellular factors of the immune system.
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http://dx.doi.org/10.1016/j.celrep.2013.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866279PMC
November 2013

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

Hyaline fibromatosis syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors.

EMBO Mol Med 2011 Apr 15;3(4):208-21. Epub 2011 Feb 15.

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

Hyaline Fibromatosis Syndrome (HFS) 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 Ig-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the Ig-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.
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http://dx.doi.org/10.1002/emmm.201100124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3377065PMC
April 2011

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

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

Hum Mutat 2009 Apr;30(4):583-9

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

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.
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http://dx.doi.org/10.1002/humu.20872DOI Listing
April 2009

Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies.

PLoS Biol 2008 Sep;6(9):e214

Department of Biochemistry, University of Geneva, Geneva, Switzerland.

After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs--an adaptor--like protein that binds membrane PtdIns3P via a FYVE motif-and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.
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http://dx.doi.org/10.1371/journal.pbio.0060214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2528051PMC
September 2008

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

Sendai virus budding in the course of an infection does not require Alix and VPS4A host factors.

Virology 2007 Aug 27;365(1):101-12. Epub 2007 Apr 27.

Department of Microbiology and Molecular Medicine, University of Geneva Medical School, CMU, 1 rue Michel Servet, 1211 Geneva 4, Switzerland.

Closing the Sendai virus C protein open reading frames (rSeV-DeltaC virus) results in the production of virus particles with highly reduced infectivity. Besides, the Sendai virus C proteins interact with Alix/AIP1 and Alix suppression negatively affects Sendai virus like particle (VLP) budding. Similarly, the Sendai virus M protein has been shown to interact with Alix. On this basis, it has been suggested that Sendai virus budding involves recruitment of the multivesicular body formation machinery. We follow, here, the production of SeV particles upon regular virus infection. We find that neither Alix suppression nor dominant negative-VPS4A expression, applied separately or in combination, affects physical or infectious virion production. This contrasts with the observed decrease of SV5 virion production upon dominant negative-VPS4A expression. Finally, we show that suppression of more than 70% of a GFP/C protein in the background of a rSeV-DeltaC virus infection has no effect either on SeV particle production or on virus particle infectivity. Our results contrast with what has been published before. Possible explanations for this discrepancy are discussed.
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http://dx.doi.org/10.1016/j.virol.2007.03.039DOI Listing
August 2007

Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival.

Cell 2006 Sep;126(6):1135-45

Department Microbiology and Molecular Medicine, University of Geneva, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland.

Many pathogenic organisms produce pore-forming toxins as virulence factors. Target cells however mount a response to such membrane damage. Here we show that toxin-induced membrane permeabilization leads to a decrease in cytoplasmic potassium, which promotes the formation of a multiprotein oligomeric innate immune complex, called the inflammasome, and the activation of caspase-1. Further, we find that when rendered proteolytic in this context caspase-1 induces the activation of the central regulators of membrane biogenesis, the Sterol Regulatory Element Binding Proteins (SREBPs), which in turn promote cell survival upon toxin challenge possibly by facilitating membrane repair. This study highlights that, in addition to its well-established role in triggering inflammation via the processing of the precursor forms of interleukins, caspase-1 has a broader role, in particular linking the intracellular ion composition to lipid metabolic pathways, membrane biogenesis, and survival.
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http://dx.doi.org/10.1016/j.cell.2006.07.033DOI Listing
September 2006

Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis.

J Cell Biol 2006 Jan 9;172(2):309-20. Epub 2006 Jan 9.

Department of Microbiology and Molecular Medicine, University of Geneva, 1211 Geneva 4, Switzerland.

The anthrax toxin is composed of three independent polypeptide chains. Successful intoxication only occurs when heptamerization of the receptor-binding polypeptide, the protective antigen (PA), allows binding of the two enzymatic subunits before endocytosis. We show that this tailored behavior is caused by two counteracting posttranslational modifications in the cytoplasmic tail of PA receptors. The receptor is palmitoylated, and this unexpectedly prevents its association with lipid rafts and, thus, its premature ubiquitination. This second modification, which is mediated by the E3 ubiquitin ligase Cbl, only occurs in rafts and is required for rapid endocytosis of the receptor. As a consequence, cells expressing palmitoylation-defective mutant receptors are less sensitive to anthrax toxin because of a lower number of surface receptors as well as premature internalization of PA without a requirement for heptamerization.
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http://dx.doi.org/10.1083/jcb.200507067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2063559PMC
January 2006

Anthrax toxin: the long and winding road that leads to the kill.

Trends Microbiol 2005 Feb;13(2):72-8

Department of Microbiology and Molecular Medicine, University of Geneva, 1 rue Michel Servet, 1211 Geneva 4, Switzerland.

The past five years have led to a tremendous increase in our molecular understanding of the mode of action of the anthrax toxin, one of the two main virulence factors produced by Bacillus anthracis. The structures of each of the three components of the toxin--lethal factor (LF), edema factor (EF) and protective antigen (PA)--have been solved not only in their monomeric forms but, depending on the subunit, in a heptameric form, bound to their substrate, co-factor or receptor. The endocytic route followed by the toxin has also been unraveled and the enzymatic mechanisms of EF and LF elucidated.
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http://dx.doi.org/10.1016/j.tim.2004.12.004DOI Listing
February 2005

Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway.

J Cell Biol 2004 Aug;166(5):645-51

Department of Microbiology and Molecular Medicine, University of Geneva, 1 rue Michel Servet, Geneva, Switzerland 1211.

The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.
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http://dx.doi.org/10.1083/jcb.200312072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172425PMC
August 2004

Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins.

Proc Natl Acad Sci U S A 2004 Jul 15;101(30):10995-1000. Epub 2004 Jul 15.

Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, 92093, USA.

Cytolytic pore-forming toxins are important for the virulence of many disease-causing bacteria. How target cells molecularly respond to these toxins and whether or not they can mount a defense are poorly understood. By using microarrays, we demonstrate that the nematode Caenorhabditis elegans responds robustly to Cry5B, a member of the pore-forming Crystal toxin family made by Bacillus thuringiensis. This genomic response is distinct from that seen with a different stressor, the heavy metal cadmium. A p38 mitogen-activated protein kinase (MAPK) kinase and a c-Jun N-terminal-like MAPK are both transcriptionally up-regulated by Cry5B. Moreover, both MAPK pathways are functionally important because elimination of either leads to animals that are (i) hypersensitive to a low, chronic dose of toxin and (ii) hypersensitive to a high, brief dose of toxin such that the animal might naturally encounter in the wild. These results extend to mammalian cells because inhibition of p38 results in the hypersensitivity of baby hamster kidney cells to aerolysin, a pore-forming toxin that targets humans. Furthermore, we identify two downstream transcriptional targets of the p38 MAPK pathway, ttm-1 and ttm-2, that are required for defense against Cry5B. Our data demonstrate that cells defend against pore-forming toxins by means of conserved MAPK pathways.
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http://dx.doi.org/10.1073/pnas.0404073101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC503732PMC
July 2004

Bacterial subversion of lipid rafts.

Curr Opin Microbiol 2004 Feb;7(1):4-10

Department of Genetics and Microbiology, University of Geneva, 1 rue Michel Servet, 1211 Geneva 4, Switzerland.

Bacteria rely on numerous basic cellular functions of their target cells to reach successful infection. The recent discovery that the plasma membrane contains specialized microdomains, called lipid rafts, with many specific functions but in particular with the ability to concentrate signaling molecules, has therefore attracted the attention of cellular microbiologists. Since then an increasing number of bacteria and their products have been shown to interact with lipid rafts to promote infection or intoxication. Here we review why certain bacteria and/or their products are attracted toward these lipid microdomains.
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http://dx.doi.org/10.1016/j.mib.2003.12.007DOI Listing
February 2004

Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process.

J Cell Biol 2003 Feb 27;160(3):321-8. Epub 2003 Jan 27.

Department of Genetics and Microbiology, University of Geneva, 1211 Geneva 4, Switzerland.

The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.
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http://dx.doi.org/10.1083/jcb.200211018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172673PMC
February 2003