Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2.

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.

Spontaneous reactivation of clusters of X-linked genes is associated with the plasticity of X-inactivation in mouse trophoblast stem cells.

Random epigenetic silencing of the X-chromosome in somatic tissues of female mammals equalizes the dosage of X-linked genes between the sexes. Unlike this form of X-inactivation that is essentially irreversible, the imprinted inactivation of the paternal X, which characterizes mouse extra-embryonic tissues, appears highly unstable in the trophoblast giant cells of the placenta. Here, we wished to determine whether such instability is already present in placental progenitor cells prior to differentiation toward lineage-specific cell types. To this end, we analyzed the behavior of a GFP transgene on the paternal X both in vivo and in trophoblast stem (TS) cells derived from the trophectoderm of XX(GFP) blastocysts. Using single-cell studies, we show that not only the GFP transgene but also a large number of endogenous genes on the paternal X are subject to orchestrated cycles of reactivation/de novo inactivation in placental progenitor cells. This reversal of silencing is associated with local losses of histone H3 lysine 27 trimethylation extending over several adjacent genes and with the topological relocation of the hypomethylated loci outside of the nuclear compartment of the inactive X. The "reactivated" state is maintained through several cell divisions. Our study suggests that this type of "metastable epigenetic" states may underlie the plasticity of TS cells and predispose specific genes to relaxed regulation in specific subtypes of placental cells.

Screening of a Drug Library Identifies Inhibitors of Cell Intoxication by CNF1.

Cytotoxic necrotizing factor 1 (CNF1) is a toxin produced by pathogenic strains of Escherichia coli responsible for extra-intestinal infections. CNF1 deamidates Rac1, thereby triggering its permanent activation and worsening inflammatory reactions. Activated Rac1 is prone to proteasomal degradation. There is no targeted therapy against CNF1, despite its clinical relevance. In this work we developed a fluorescent cell-based immunoassay to screen for inhibitors of CNF1-induced Rac1 degradation among 1120 mostly approved drugs. Eleven compounds were found to prevent CNF1-induced Rac1 degradation, and five also showed a protective effect against CNF1-induced multinucleation. Finally, lasalocid, monensin, bepridil, and amodiaquine protected cells from both diphtheria toxin and CNF1 challenges. These data highlight the potential for drug repurposing to fight several bacterial infections and Rac1-based diseases.

Creation of intercellular bonds by anchoring protein ligands to membranes using the diphtheria toxin T domain.

We describe the creation of cell adhesion mediated by cell surface engineering. The Flt3-ligand was fused to a membrane anchor made of the diphtheria toxin translocation domain. The fusion protein was attached to the surface of a cell by an acid pulse. Contact with another cell expressing the receptor Flt3 lead to its activation. This activity involved direct cell-cell contact. A mean force of 20 nN was needed to separate functionalized cells after 5 min of contact. Overall, we showed that it is possible to promote specific cell-cell adhesion by attaching protein ligands at the surface of cells.

Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes.

The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH.

Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC.

Correction: The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH.

[This corrects the article DOI: 10.1371/journal.pone.0153401.].

Amyloid fibrils formed by the programmed cell death regulator Bcl-xL.

The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis. Bcl-xL forms aggregates in three states, micelles, native-like fibrils, and amyloid fibers, and their biophysical characterization has been performed in detail. Bcl-xL remains in its native state within micelles and native-like fibrils, and our results suggest that native-like fibrils are formed by the association of micelles. Formation of amyloid structures, that is, nonnative intermolecular ß-sheets, is favored by the proximity of proteins within fibrils at the expense of the Bcl-xL native structure. Finally, we provide evidence of a direct relationship between the amyloid character of the fibers and the tertiary-structure stability of the native Bcl-xL. The potential causality between the accumulation of Bcl-xL into amyloid deposits and abnormal apoptosis during neurodegenerative diseases is discussed.

Solution and membrane-bound chaperone activity of the diphtheria toxin translocation domain towards the catalytic domain.

During cell intoxication by diphtheria toxin, endosome acidification triggers the translocation of the catalytic (C) domain into the cytoplasm. This event is mediated by the translocation (T) domain of the toxin. Previous work suggested that the T domain acts as a chaperone for the C domain during membrane penetration of the toxin. Using partitioning experiments with lipid vesicles, fluorescence spectroscopy, and a lipid vesicle leakage assay, we characterized the dominant behavior of the T domain over the C domain during the successive steps by which these domains interact with a membrane upon acidification: partial unfolding in solution and during membrane binding, and then structural rearrangement during penetration into the membrane. To this end, we compared, for each domain, isolated or linked together in a CT protein (the toxin lacking the receptor-binding domain), each of these steps. The behavior of the T domain is marginally modified by the presence or absence of the C domain, whereas that of the C domain is greatly affected by the presence of the T domain . All of the steps leading to membrane penetration of the C domain are triggered at higher pH by the T domain , by 0.5-1.6 pH units. The T domain stabilizes the partially folded states of the C domain corresponding to each step of the process. The results unambiguously demonstrate that the T domain acts as a specialized pH-dependent chaperone for the C domain. Interestingly, this chaperone activity acts on very different states of the protein: in solution, membrane-bound, and membrane-inserted.

Accessibility changes within diphtheria toxin T domain upon membrane penetration probed by hydrogen exchange and mass spectrometry.

The translocation domain of diphtheria toxin inserts in membrane and becomes functional when the pH inside endosomes is acid. At that stage, the domain is in a partially folded state; this prevents the use of high-resolution methods for the characterization of its functional structure. On that purpose, we report here the use of hydrogen/deuterium exchange experiments coupled to mass spectrometry. The conformation changes during the different steps of insertion into lipid bilayer are monitored with a resolution of few residues. Three parts of the translocation domain can be distinguished. With a high protection against exchange, the C-terminal hydrophobic helical hairpin is embedded in the membrane. Despite a lower protection, a significant effect in the presence of lipid vesicles shows that the N-terminal part is in interaction with the membrane interface. The sensitivity to the ionic strength indicates that electrostatic interactions are important for the binding. The middle part of the domain has an intermediate protection; this suggests that this part of the domain can be embedded within the membrane but remains quite dynamic. These results provide unprecedented insight into the structure reorganization of the protein to go from a soluble state to a membrane-inserted one.

Accessibility changes within diphtheria toxin T domain when in the functional molten globule state, as determined using hydrogen/deuterium exchange measurements.

The translocation domain (T domain) of diphtheria toxin adopts a partially folded state, the so-called molten globule state, to become functional at acidic pH. We compared, using hydrogen/deuterium exchange experiments associated with MS, the structures of the T domain in its soluble folded state at neutral pH and in its functional molten globule state at acidic pH. In the native state, the alpha-helices TH5 and TH8 are identified as the core of the domain. Based on the high-resolution structure of the T domain, we propose that TH8 is highly protected because it is buried within the native structure. According to the same structure, TH5 is partly accessible at the surface of the T domain. We propose that its high protection is caused by the formation of dimers. Within the molten globule state, high protection is still observed within the helical hairpin TH8-TH9, which is responsible for the insertion of the T domain into the membrane. In the absence of the lipid bilayer, this hydrophobic part of the domain self-assembles, leading to the formation of oligomers. Overall, hydrogen/deuterium-exchange measurements allow the analysis of interaction contacts within small oligomers made of partially folded proteins. Such information, together with crystal structure data, are particularly valuable for using to analyze the self-assembly of proteins.

Behavior of the N-terminal helices of the diphtheria toxin T domain during the successive steps of membrane interaction.

During intoxication of a cell, the translocation (T) domain of the diphtheria toxin helps the passage of the catalytic domain across the membrane of the endosome into the cytoplasm. We have investigated the behavior of the N-terminal region of the T domain during the successive steps of its interaction with membranes at acidic pH using tryptophan fluorescence, its quenching by brominated lipids, and trypsin digestion. The change in the environment of this region was monitored using mutant W281F carrying a single native tryptophan at position 206 at the tip of helix TH1. The intrinsic propensity to interact with the membrane of each helix of the N-terminus of the T domain, TH1, TH2, TH3, and TH4, was also studied using synthetic peptides. We showed the N-terminal region of the T domain was not involved in the binding of the domain to the membrane, which occurred at pH 6 mainly through hydrophobic effects. At that stage of the interaction, the N-terminal region remained strongly solvated. Further acidification eliminated repulsive electrostatic interactions between this region and the membrane, allowing its penetration into the membrane by attractive electrostatic interactions and hydrophobic effects. The peptide study indicated the nature of forces contributing to membrane penetration. Overall, the data suggested that the acidic pH found in the endosome not only triggers the formation of the molten globule state of the T domain required for membrane interaction but also governs a progressive penetration of the N-terminal part of the T domain in the membrane. We propose that these physicochemical properties are necessary for the translocation of the catalytic domain.

Concerted protonation of key histidines triggers membrane interaction of the diphtheria toxin T domain.

The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His(223)-His(257) and His(251) are the most sensitive triggers for the formation of the molten globule state in solution, whereas His(322)-His(323) and His(251) are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.

Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation.

X-chromosome inactivation (XCI) is highly dynamic during early mouse embryogenesis and strictly depends on the Xist noncoding RNA. The regulation of Xist and its antisense partner Tsix remains however poorly understood. We provide here the first evidence of transcriptional control of Xist expression. We show that RNA polymerase II (RNAPolII) preinitiation complex recruitment and H3 Lys 4 (H3-K4) methylation at the Xist promoter form the basis of the Xist expression profiles that drives both imprinted and random XCI. In embryonic stem (ES) cells, which are derived from the inner cell mass where imprinted XCI is reversed and both Xs are active, we show that Xist is repressed at the level of preinitiation complex (PIC) recruitment. We further demonstrate that Tsix, although highly transcribed in ES cells, is not itself responsible for the transcriptional down-regulation of Xist. Rather, Tsix induces efficient H3-K4 methylation over the entire Xist/Tsix unit. We suggest that chromatin remodeling of the Xist locus induced by biallelic Tsix transcription renders both Xist loci epigenetically equivalent and equally competent for transcription. In this model, Tsix, by resetting the epigenetic state of the Xist/Tsix locus, mediates the transition from imprinted to random XCI.