Monalysin, a Novel ß-Pore-Forming Toxin from the Drosophila Pathogen Pseudomonas entomophila, Contributes to Host Intestinal Damage and Lethality

Pseudomonas entomophila is an entomopathogenic bacterium that infects and kills Drosophila. P. entomophila pathogenicity is linked to its ability to cause irreversible damages to the Drosophila gut, preventing epithelium renewal and repair. Here we report the identification of a novel pore-forming toxin (PFT), Monalysin, which contributes to the virulence of P. entomophila against Drosophila. Our data show that Monalysin requires N-terminal cleavage to become fully active, forms oligomers in vitro, and induces pore-formation in artificial lipid membranes. The prediction of the secondary structure of the membrane-spanning domain indicates that Monalysin is a PFT of the ß-type. The expression of Monalysin is regulated by both the GacS/GacA two-component system and the Pvf regulator, two signaling systems that control P. entomophila pathogenicity. In addition, AprA, a metallo-protease secreted by P. entomophila, can induce the rapid cleavage of pro-Monalysin into its active form. Reduced cell death is observed upon infection with a mutant deficient in Monalysin production showing that Monalysin plays a role in P. entomophila ability to induce intestinal cell damages, which is consistent with its activity as a PFT. Our study together with the well-established action of Bacillus thuringiensis Cry toxins suggests that production of PFTs is a common strategy of entomopathogens to disrupt insect gut homeostasis

Functional analysis of the yeast "Saccharomyces cerevisiae" Gpi8 protein and characterization of the purified GPI-transamidase complex

Dans le monde des Eucaryotes inférieurs ou supérieurs, de nombreuses glycoprotéines sont ancrées dans leur membrane plasmique par l’intermediaire d’une ancre GPI (glycosylphosphatidylinositol). Cette modification post-traductionnelle est nécessaire pour la croissance et le développement de la levure Saccharomyces cerevisiae. Chez cet organisme, environ 70 cadres ouverts de lecture prédisent des proteins ancrées GPI, la majorité étant des glycoprotéines de la paroi cellulaire. Les précurseurs des protéines ancrées GPI possèdent du côté N-terminal un signal d’importation dans le reticulum endoplasmique (RE), et du côté Cterminal un signal d’ancrage GPI qui est nécessaire et suffisant pour l’addition d’une ancre GPI. Le signal d’ancrage GPI (composé d’un domaine C-terminal hydrophobique séparé du site de clivage par une petite region hydrophilique) est reconnu et enlevé par une transamidase spécifique qui remplace ce signal par une ancre GPI préformée. Cette transférase à ancres GPI fonctionne telle une transamidase, à savoir qu’elle retire le signal d’ancrage et le remplace conjointement par une ancre GPI préformée. Les cellules déficientes pour cette transamidase accumulent des ancres GPI complètes (appelées lipides CP2), ainsi que des précurseurs de protéines conservant leur signal d’ancrage GPI. Ce phénotype est observé chez deux mutants de la levure, gaa1 et gpi8. Gpi8p et Gaa1p sont deux composantes essentielles de la transamidase responsable du transfert des ancres GPI sur les protéines nouvellement synthétisées. GPI8 est essentiel et code pour une proteine membranaire de type I de 50 kDa, caractérisée par un domaine transmembranaire absent chez C. elegans et chez certains protozoaires. Gpi8p partage 25-28% d’homologie de sequence avec une famille de protéases à cystéines (famille C13) qui présente une activité enzymatique propre aux transamidases. Les résidus Cys199 et His156 du site actif, identifiés après comparaison avec le site actif des caspases, sont essentiels; leur mutation en Ala donne lieu à des allèles GPI8 non fonctionnels. La proteine Gpi8p de la levure est totalement fonctionnelle sans son domaine C-terminal transmembranaire. Ce domaine est cependant important dans la retention de la protéine Gpi8 dans le RE. Une proteine Gpi8 pure, obtenue après purification de la partie soluble de Gpi8p, ne présente aucune activité de transamidase, testée in vitro sur des peptides substrats. GAA1 est essentiel et code pour une protéine du RE de 68 kDa caractérisée par un large domaine luminal hydrophilique, et par plusieurs domaines transmembranaires prolongés en C-terminal par un signal cytosolique de retour vers le RE. La solubilisation dans 1.5 % de digitonine, suivie d’une séparation par électrophorèse sur gel natif de polyacrylamide, montre que Gpi8p est localisée dans des complexes protéiques de 430 à 650 kDa. Ces complexes ont été purifiés par des techniques de chromatographie d’affinité et les sous-unités analysées par des techniques de spectrophotometrie de masse. Ces analyses ont revelé la presence dans ces complexes de Gaa1p, Gpi8p, et d’une nouvelle protéine, Gpi16p (YHR188c). Gpi16p est une protéine transmembranaire, N-glycosylée, et essentielle. La protéine est située dans la partie luminale du RE, caractérisée par un domaine C-terminal transmembranaire prolongé par une petite partie cytosolique affichant un signal de retour vers le RE. La déplétion de Gpi16p résulte en l’accumulation dans le RE de lipides CP2, et de précurseurs protéiques non maturés. Gpi8p et Gpi16p sont instables si l’une ou l’autre des protéines est absente. Lors d’une surexpression de Gpi8p, la majorité des protéines se trouvant en dehors des complexes de 430 à 650 kDa est instable, puis rapidement dégradée. La surexpression de Gpi8p ne peut en aucun cas compenser le manque de Gpi16p. Des homologues de Gpi16p existent dans tous les organismes Eucaryotes. Le complexe de la transamidase ne se trouve associé, ni avec le complexe Sec61 requis pour le transfert des proteines dans le RE, ni avec le complexe de l’ oligosaccharyltransferase requis dans la Nglycosylation. Lorsque les substrats de la transamidase, à savoir les précurseurs protéiques ainsi que les lipides CP2, sont dépletés, le complexe enzymatique reste stable. Récement, l’identification de Gpi17p, une autre nouvelle sous-unité du complexe de la transamidase à GPI a été rapportée (Ohishi et al., 2001). Gpi17p est une protéine transmembranaire, Nglycosylée, et essentielle. La protéine est située dans la partie luminale du RE, et présente deux domaines transmembranaires aux extremités N- et C-terminaux. La depletion de Gpi17p entraine une accumulation de lipides GPI complets CP2. L’interaction entre les parties hydrophiliques et luminales de chacune des composantes du complexe de la transamidase (Gpi8p, Gaa1p, Gpi16p, et Gpi17p) a été étudiée par l’intermédiaire du système de double hybride basé sur le promoteur GAL4. Un modèle de l’organisation macromoleculaire du complexe de la transamidase est ici proposé.Many glycoproteins of lower and higher eucaryotes are attached to the plasma membrane by means of a glycosylphosphatidylinositol (GPI). GPI anchoring of proteins is essential for the growth of Saccharomyces cerevisiae . S.cerevisiae contains about 70 open reading frames predicting GPI proteins and many of these have been found to be cell wall glycoproteins. The precursors of GPI anchored proteins have a classical signal sequence for import into the endoplasmic reticulum at their N-terminus and a GPI anchoring signal at their C-terminus which is necessary and sufficient to direct GPI addition. The C-terminal GPI anchoring signal (composed of a C-terminal hydrophobic domain separated by a short hydrophilic spacer from the cleavage/attachment site) is recognized and removed by a GPI transamidase, which replaces it by a preformed GPI. The GPI transferase is believed to act as a transamidase, i.e. to jointly remove the GPI anchoring signal and transfer the preformed GPI. Transamidasedeficient cells are expected to accumulate complete GPI’s as well as GPI precursor proteins retaining the GPI anchoring signal. This phenotype is exhibited by two yeast mutants, gaa1 and gpi8. Gpi8p and Gaa1p are essential components of the GPI transamidase which adds GPIs to newly synthesized proteins. GPI8 is essential and encodes a 50 kDa type I ER membrane protein with a single membrane spanning domain (MSD), which is entirely lacking in C. elegans and some protozoan Gpi8p. Gpi8p has 25-28% homology to a family of cysteine proteinase (C13 family), one of which is able to act as a transamidase. The Cys and His residues, predicted to be active sites by sequence comparison with caspases indeed are essential and their mutation to Ala yields non-functional GPI8 alleles. Yeast Gpi8p is fully functional without its C-terminal membrane spanning domain. This domain however helps to retain Gpi8p in the ER. Purification of the water soluble part of Gpi8p to homogeneity yields pure Gpi8p that has no detectable transamidase activity towards suitable peptides in vitro. GAA1 is essential and encodes a 68 kDa ER protein with a large, hydrophilic, lumenal domain, followed by several MSDs and a cytosolic ER retrieval signal on its extreme Cterminus. After solubilization in 1.5 % digitonin and separation by blue native polyacrylamide gel electrophoresis, Gpi8p is found in 430 to 650 kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, it has a single C-terminal transmembrane domain and a small Cterminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430 to 650 kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact. Recently, the identification of Gpi17p, another new component of the GPI transamidase complex was reported (Ohishi et al., 2001). Gpi17p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and two transmembrane domains are predicted near the N- and C-termini. Depletion of Gpi17p results in the accumulation of the complete GPI lipid CP2. The interaction of the hydrophilic, lumenal parts of each component of the transamidase complex, Gpi8p, Gaa1p, Gpi16p, and Gpi17p, with each other, was studied in a GAL4-based two hybrid system. From thus, a model of the macromolecular organization of the transamidase complex is proposed

SwissPalm: Protein Palmitoylation database

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. © 2015 Blanc M et al

Sliding doors: clathrin-coated pits or caveolae?

Some cell-surface receptors activate downstream signal transduction pathways not only from the cell surface but also from endosomes, suggesting that signalling pathways can be regulated by compartmentalization. A further twist is that different internalization routes seem to predetermine whether transforming growth factor β (TGF-β) receptors will trigger a signalling response or be degraded

USP19_b stimulates the cytotoxicities of polyQ-expanded Atx3_100Q (A) and Htt-N171_100Q (B).

<p>Here Htt-N171_100Q refers to the N-terminal 171-residue fragment of Htt with 100Q. HEK 293T cells were co-transfected with the plasmids as indicated. After 48-hour culture, the cells were subjected to CytoTox-ONE^™ assay. Data were statistically analyzed with one-way ANOVA and presented as Mean ± SD (n = 6). *, p < 0.05; **, p < 0.01; N.S., no significance.</p

USP19_b associates with HSP90 through CS domains.

<p><b>A</b>, Co-IP experiment showing interaction of USP19_b with HSP90. HEK 293T cells were co-transfected with HA-USP19_b and HSP90-Myc plasmids, and then the cell lysates were subjected to immunoprecipitation with protein A/G-conjugated anti-Myc antibody. The control lane is in the presence of only protein A/G. <b>B</b>, Immunoprecipitation with the endogenous HSP90 and CHIP by USP19_b. HEK 293T cells were transiently transfected with HA-USP19_b expression plasmid. <b>C</b>, GST pull-down experiment showing direct interaction between CS1 (residues 75–209) or CS2 (273–393) domain of USP19_b and HSP90. GST protein was set as a control. The arrow indicates the band of HSP90. <b>D</b>, Sequence alignment of the CS domains from USP19 (<i>Homo Sapiens</i>) and Sgt1a (<i>Arabidopsis</i>). The conserved residues that are putatively important to HSP90 binding were selected for mutation. <b>E</b>, Co-IP experiment of USP19_b or its CS-domain mutants with HSP90. CS1M, K176A/Q178A in the CS1 domain; CS2M, Y290A/Y302A in the CS2 domain; CS12M, double-domain mutant. HSP90 was Myc tagged, while USP19_b and its mutants were HA tagged.</p

Inhibition of HSP90 down-regulates the protein levels and aggregates of Atx3_100Q and Htt-N552_100Q.

<p><b>A</b> and <b>B</b>, Effects of HSP90 inhibitor on the protein levels of Atx3_100Q (<b>A</b>) and Htt-N552_100Q (<b>B</b>). FLAG-tagged Atx3_100Q or Htt-N552_100Q was transfected into HEK 293T cells, and then the cells were treated with different doses of 17-AAG for 6 hrs (DMSO as a control). About 48 hrs after transfection, the cells were harvested and lysed for Western blotting. <b>C</b> and <b>D</b>, Effects of HSP90 inhibitor on the SDS-resistant aggregates of Atx3_100Q (<b>C</b>) and Htt-N552_100Q (<b>D</b>) by filter trap analysis. Data were quantitated with relative band intensities and presented as Mean ± SEM (n = 3).</p

USP19_b increases the protein levels of Atx3_100Q and Htt-N552_100Q.

<p><b>A</b>, Domain architecture and sequence alignment of USP19 isoforms. USP19 contains two CHORD-SGT1 domains (namely CS1 and CS2) at its N-terminus and a large USP domain. Instead of a transmembrane domain in USP19_a, there is a relatively hydrophilic region and an EEVD motif in the C-terminus of USP19_b. <b>B</b> and <b>C</b>, Effects of USP19_b on the protein levels of Atx3_100Q (<b>B</b>) and Htt-N552_100Q (<b>C</b>). HEK 293T cells were transfected with equal amount of FLAG-tagged Atx3_100Q or Htt-N552_100Q and vector, HA-USP19_b or its C506S mutant, and 48 hrs later, the cells were harvested and lysed for Western blotting with the indicated antibodies. <b>D,</b> Effect of USP19_b on the protein level of endogenous Atx3. <b>E</b>, Effect of USP5 on the protein level of Atx3_100Q. FLAG-tagged Atx3_100Q was co-transfected with HA-USP5 or its active-site mutant (C335A) into HEK 293T cells. <b>F</b> and <b>G</b>, Knockdown of USP19 reduces the protein level of Atx3_100Q (<b>F</b>) or Htt-N552_100Q (<b>G</b>) in HEK 293T cells. Cells were transfected with FLAG-tagged Atx3_100Q or Htt-N552_100Q and USP19 siRNA. After 72 hrs, the cells were harvested and the lysates were subjected to Western blotting with the indicated antibodies. Ctrl, VSVG siRNA; 1#, 3#, two siRNAs against USP19. The band intensities were quantitated by using <i>Scion</i> Image. Data were normalized to mock transfected with vector or control siRNA, and statistically analyzed with one-way ANOVA and presented as Mean ± SEM (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., no significance.</p