Daniel Gillet

Inhibition of Retrograde Transport Protects Mice from Lethal Ricin Challenge

Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.

Interaction between the two subdomains of the C-terminal part of the botulinum neurotoxin A is essential for the generation of protective antibodies.

The botulinum neurotoxin A C‐terminal fragment (Hc), which mediates the binding of the toxin to neuronal cell surface receptors, comprises two subdomains, Hc‐N (amino acids 873–1095) and Hc‐C (amino acids 1096–1296). In order to define the minimal fragment of Hc carrying protective antigenic properties, Hc, Hc‐N and Hc‐C have been produced as recombinant proteins in Escherichia coli, and have been tested for their antigenicity in mouse protection assays. Hc, Hc‐N and Hc‐C induced similar antibody levels as shown by ELISA. However, a single immunization with Hc (10 μg) fully protected mice challenged with 103 mouse lethal dose 50 of toxin, whereas Hc‐N, Hc‐C, or Hc‐N plus Hc‐C did not give any protection. Triple immunizations with Hc‐N or Hc‐C were necessary to induce a higher level of protection. Circular dichroism and fluorescence studies showed that the isolated subdomains were folded and stable. However, an intense near‐UV dichroic signal was only observed in the Hc spectrum, revealing a highly structured interface between both subdomains. Taken together, the results show that the generation of protective antibodies requires the whole Hc domain and especially the native structure of the interfacial region between Hc‐N and Hc‐C.

One NY-ESO-1-Derived Epitope That Promiscuously Binds to Multiple HLA-DR and HLA-DP4 Molecules and Stimulates Autologous CD4+ T Cells from Patients with NY-ESO-1-Expressing Melanoma

NY-ESO-1 is expressed by a broad range of human tumors and is often recognized by Abs in the sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and class II-restricted tumor epitopes recognized by T lymphocytes. In this study we report one novel pan-MHC class II-restricted peptide sequence, NY-ESO-1 87–111, that is capable of binding to multiple HLA-DR and HLA-DP4 molecules, including HLA-DRB1*0101, 0401, 0701, and 1101 and HLA-DPB1*0401 and 0402 molecules. We also demonstrate that peptide NY-ESO-1 87–111 stimulates Th1-type and Th-2/Th0-type CD4+ T cells and clones when presented in the context of these HLA-DR and HLA-DP4 molecules. Both bulk CD4+ T cells and CD4+ T cell clones were capable of recognizing not only peptide-pulsed APCs, but also autologous dendritic cells, either loaded with the NY-ESO-1 protein or transfected with NY-ESO-1 cDNAs. Using IFN-γ and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4+ T cells recognizing peptide NY-ESO-1 87–111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87–111 in the large number of cancer patients with NY-ESO-1-expressing tumors.

Membrane Interaction of botulinum neurotoxin A translocation (T) domain. The belt region is a regulatory loop for membrane interaction.

The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.

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 His223-His257 and His251 are the most sensitive triggers for the formation of the molten globule state in solution, whereas His322-His323 and His251 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.

N-Methyldihydroquinazolinone Derivatives of Retro-2 with Enhanced Efficacy against Shiga Toxin

The Retro-2 molecule protects cells against Shiga toxins by specifically blocking retrograde transport from early endosomes to the trans-Golgi network. A SAR study has been carried out to identify more potent compounds. Cyclization and modifications of Retro-2 led to a compound with roughly 100-fold improvement of the EC50 against Shiga toxin cytotoxicity measured in a cell protein synthesis assay. We also demonstrated that only one enantiomer of the dihydroquinazolinone reported herein is bioactive.

Deciphering Membrane Insertion of the Diphtheria Toxin T Domain by Specular Neutron Reflectometry and Solid-State NMR Spectroscopy

Insertion and translocation of soluble proteins into and across biological membranes are involved in many physiological and pathological processes, but remain poorly understood. Here, we describe the pH-dependent membrane insertion of the diphtheria toxin T domain in lipid bilayers by specular neutron reflectometry and solid-state NMR spectroscopy. We gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. At pH 6, the native tertiary structure of the T domain unfolds, allowing its binding to the membrane. The membrane-bound state is characterized by a localization of the C-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. In contrast, the amphiphilic N-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. At pH 4, repulsive interactions vanish; the N-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. The C-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. These helices do not adopt a transmembrane orientation. Interestingly, the T domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. We propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane.

Inhibitors of the Cellular Trafficking of Ricin

Throughout the last decade, efforts to identify and develop effective inhibitors of the ricin toxin have focused on targeting its N-glycosidase activity. Alternatively, molecules disrupting intracellular trafficking have been shown to block ricin toxicity. Several research teams have recently developed high-throughput phenotypic screens for small molecules acting on the intracellular targets required for entry of ricin into cells. These screens have identified inhibitory compounds that can protect cells, and sometimes even animals against ricin. We review these newly discovered cellular inhibitors of ricin intoxication, discuss the advantages and drawbacks of chemical-genetics approaches, and address the issues to be resolved so that the therapeutic development of these small-molecule compounds can progress.

Alteration of the tertiary structure of the major bee venom allergen Api m 1 by multiple mutations is concomitant with low IgE reactivity

We have engineered a recombinant form of the major bee venom allergen (Api m 1) with the final goal of reducing its IgE reactivity. This molecule (Api mut) contains 24 mutations and one deletion of 10 amino acids. The successive introduction of these sequence modifications led to a progressive loss of specific IgE and IgG reactivity and did not reveal any immunodominant epitopes. However, Api mut exhibited a clear loss of reactivity for Api m 1‐specific IgE and IgG. Injection of Api mut into mice induced specific antibody production. This humoral response was as high as that induced by the Api m 1 but the cross‐reactivity of the antibodies was weak. As inferred by far UV circular dichroism, this mutant was correctly folded. However, near UV circular dichroism and denaturation curves of Api mut showed that it exhibits a dynamic tertiary structure and that it is a highly flexible molecule. Finally, as all the sequence modifications have been introduced outside the human and murine T cell epitope regions, we investigated its T cell properties in mice. We showed that Api mut‐specific T lymphocytes induced in vivo were stimulated in vitro by both proteins. These data provide new insights in the design of hypoallergenic molecules.

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 α‐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.