A binding motif for Hsp90 in the A chains of ADP‐ribosylating toxins that move from the endoplasmic reticulum to the cytosol

Cholera toxin (Ctx) is an AB‐type protein toxin that acts as an adenosine diphosphate (ADP)‐ribosyltransferase to disrupt intracellular signalling in the target cell. It moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic CtxA1 subunit then dissociates from the rest of the toxin, unfolds, and activates the ER‐associated degradation system for export to the cytosol. Translocation occurs through an unusual ratchet mechanism in which the cytosolic chaperone Hsp90 couples CtxA1 refolding with CtxA1 extraction from the ER. Here, we report that Hsp90 recognises two peptide sequences from CtxA1: an N‐terminal RPPDEI sequence (residues 11–16) and an LDIAPA sequence in the C‐terminal region (residues 153–158) of the 192 amino acid protein. Peptides containing either sequence effectively blocked Hsp90 binding to full‐length CtxA1. Both sequences were necessary for the ER‐to‐cytosol export of CtxA1. Mutagenesis studies further demonstrated that the RPP residues in the RPPDEI motif are required for CtxA1 translocation to the cytosol. The LDIAPA sequence is unique to CtxA1, but we identified an RPPDEI‐like motif at the N‐ or C‐termini of the A chains from four other ER‐translocating toxins that act as ADP‐ribosyltransferases: pertussis toxin, Escherichia coli heat‐labile toxin, Pseudomonas aeruginosa exotoxin A, and Salmonella enterica serovar Typhimurium ADP‐ribosylating toxin. Hsp90 plays a functional role in the intoxication process for most, if not all, of these toxins. Our work has established a defined RPPDEI binding motif for Hsp90 that is required for the ER‐to‐cytosol export of CtxA1 and possibly other toxin A chains as well.     

Photorhabdus luminescens Tc toxin is inhibited by the protease inhibitor MG132 and activated by protease cleavage resulting in increased binding to target cells

Photorhabdus luminescens Tc toxins consist of the cell‐binding component TcA, the linker component TcB, and the enzyme component TcC. TccC3, a specific isoform of TcC, ADP‐ribosylates actin and causes redistribution of the actin cytoskeleton. TccC5, another isoform of TcC, ADP‐ribosylates and activates Rho proteins. Here, we report that the proteasome inhibitor MG132 blocks the intoxication of cells by Tc toxin. The inhibitory effect of MG132 was not observed, when the ADP‐ribosyltransferase domain of the TcC component was introduced into target cells by protective antigen, which is the binding and delivery component of anthrax toxin. Additionally, MG132 affected neither pore formation by TcA in artificial membranes nor binding of the toxin to cells. Furthermore, the in vitro ADP‐ribosylation of actin by the enzyme domain of TccC3 was not affected by MG132. Similar to MG132, several calpain inhibitors blocked the action of the Tc toxin. Proteolytic cleavage of the binding component TcA induced by P. luminescens protease PrtA1 or by collagenase largely increased the toxicity of the Tc toxin. MG132 exhibited no inhibitory effect on the cleaved TcA component. Moreover, binding of TcA to target cells was largely increased after cleavage. The data indicate that Tc toxin is activated by proteolytic processing of the TcA component, resulting in increased receptor binding. Toxin processing is probably inhibited by MG132.     

Actin ADP‐ribosylation at Threonine148 by Photorhabdus luminescens toxin TccC3 induces aggregation of intracellular F‐actin

Intoxication of eukaryotic cells by Photorhabdus luminescens toxin TccC3 induces cell rounding and detachment from the substratum within a few hours and compromises a number of cell functions like phagocytosis. Here, we used morphological and biochemical procedures to analyse the mechanism of TccC3 intoxication. Life imaging of TccC3‐intoxicated HeLa cells transfected with AcGFP‐actin shows condensation of F‐actin into large aggregates. Life cell total internal reflection fluorescence (TIRF) microscopy of identically treated HeLa cells confirmed the formation of actin aggregates but also disassembly of F‐actin stress fibres. Recombinant TccC3 toxin ADP‐ribosylates purified skeletal and non‐muscle actin at threonine148 leading to a strong propensity to polymerize and F‐actin bundle formation as shown by TIRF and electron microscopy. Native gel electrophoresis shows strongly reduced binding of Thr148‐ADP‐ribosylated actin to the severing proteins gelsolin and its fragments G1 and G1–3, and to ADF/cofilin. Complexation of actin with these proteins inhibits its ADP‐ribosylation. TIRF microscopy demonstrates rapid polymerization of Thr148‐ADP‐ribosylated actin to curled F‐actin bundles even in the presence of thymosin β4, gelsolin or G1–3. Thr148‐ADP‐ribosylated F‐actin cannot be depolymerized by gelsolin or G1–3 as verified by TIRF, co‐sedimentation and electron microscopy and shows reduced treadmilling as indicated by a lack of stimulation of its ATPase activity after addition of cofilin‐1.     

New insights into the mode of action of the actin ADP-ribosylating virulence factors Salmonella enterica SpvB and Clostridium botulinum C2 toxin

The C2 toxin from Clostridium botulinum represents the prototype of the family of binary actin-ADP-ribosylating toxins. These toxins covalently transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD(+)) onto arginine-177 of actin in the cytosol of eukaryotic cells resulting in depolymerization of actin filaments and cell rounding. The C2 toxin consists of two non-linked proteins, the enzyme component C2I and the binding and translocation component C2II, which delivers C2I into host cells. The ADP-ribosyltransferase SpvB from Salmonella enterica also modifies actin, but is delivered into the host cell cytosol from intracellular growing Salmonella, most likely via type-III-secretion. We characterized the mode of action of SpvB in comparison to C2 toxin in vitro and in intact cells. We identified arginine-177 as the target for SpvB-catalyzed mono-ADP-ribosylation of actin. To compare the cellular responses following modification of actin by SpvB or by the binary toxins without the influence of other Salmonella virulence factors, we constructed a cell-permeable fusion toxin to deliver the catalytic domain of SpvB (C/SpvB) into the cytosol of target cells. This review summarizes recent findings of research on the actin ADP-ribosylating toxins regarding their cellular uptake, molecular mode of action and the cellular consequences following ADP-ribosylation of actin.     

A cell-permeable fusion toxin as a tool to study the consequences of actin-ADP-ribosylation caused by the salmonella enterica virulence factor SpvB in intact cells

The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N- and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.     

The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol

Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.     

An ADP‐ribosyltransferase Alt of bacteriophage T4 negatively regulates the Escherichia coli MazF toxin of a toxin–antitoxin module

Prokaryotic toxin–antitoxin (TA) systems are linked to many roles in cell physiology, such as plasmid maintenance, stress response, persistence and protection from phage infection, and the activities of toxins are tightly regulated. Here, we describe a novel regulatory mechanism for a toxin of Escherichia coli TA systems. The MazF toxin of MazE‐MazF, which is one of the best characterized type II TA systems, was modified immediately after infection with bacteriophage T4. Mass spectrometry demonstrated that the molecular weight of this modification was 542 Da, corresponding to a mono‐ADP‐ribosylation. This modification disappeared in cells infected with T4 phage lacking Alt, which is one of three ADP‐ribosyltransferases encoded by T4 phage and is injected together with phage DNA upon infection. In vivo and in vitro analyses confirmed that T4 Alt ADP‐ribosylated MazF at an arginine residue at position 4. Finally, the ADP‐ribosylation of MazF by Alt resulted in the reduction of MazF RNA cleavage activity in vitro, suggesting that it may function to inactivate MazF during T4 infection. This is the first example of the chemical modification of an E. coli toxin in TA systems to regulate activity.     

Co- and Post-translocation Roles for HSP90 in Cholera Intoxication

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the rest of the toxin. CTA1 then unfolds and passes through an ER translocon pore to reach its cytosolic target. Due to its intrinsic instability, cytosolic CTA1 must be refolded to achieve an active conformation. The cytosolic chaperone Hsp90 is involved with the ER to cytosol export of CTA1, but the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Moreover, potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 have not been explored. Here, we show by isotope-edited Fourier transform infrared spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and refolding of CTA1 by Hsp90 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allows disordered CTA1 to attain an active state, which is further enhanced by ADP-ribosylation factor 6, a host cofactor for CTA1. Our data indicate CTA1 translocation involves a process that couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. The molecular basis for toxin translocation elucidated in this study may also apply to several ADP-ribosylating toxins that move from the endosomes to the cytosol in an Hsp90-dependent process.     

The proton‐motive force is required for translocation of CDI toxins across the inner membrane of target bacteria

Contact‐dependent growth inhibition (CDI) is a mode of bacterial competition orchestrated by the CdiB/CdiA family of two‐partner secretion proteins. The CdiA effector extends from the surface of CDI⁺ inhibitor cells, binds to receptors on neighbouring bacteria and delivers a toxin domain derived from its C‐terminal region (CdiA‐CT). Here, we show that CdiA‐CT toxin translocation requires the proton‐motive force (pmf) within target bacteria. The pmf is also critical for the translocation of colicin toxins, which exploit the energized Ton and Tol systems to cross the outer membrane. However, CdiA‐CT translocation is clearly distinct from known colicin‐import pathways because ΔtolA ΔtonB target cells are fully sensitive to CDI. Moreover, we provide evidence that CdiA‐CT toxins can be transferred into the periplasm of de‐energized target bacteria, indicating that transport across the outer membrane is independent of the pmf. Remarkably, CDI toxins transferred under de‐energized conditions remain competent to enter the target‐cell cytoplasm once the pmf is restored. Collectively, these results indicate that outer‐ and inner‐membrane translocation steps can be uncoupled, and that the pmf is required for CDI toxin transport from the periplasm to the target‐cell cytoplasm.     

FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 inhibits membrane translocation of the toxin in mammalian cells

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I. C2IIa delivers C2I into the cytosol of eukaryotic target cells where C2I ADP-ribosylates actin. After receptor-mediated endocytosis of the C2IIa/C2I complex, C2IIa forms pores in membranes of acidified early endosomes and unfolded C2I translocates through the pores into the cytosol. Membrane translocation of C2I is facilitated by the activities of host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase (PPIase) cyclophilin A. Here, we demonstrated that Hsp90 co-precipitates with C2I from lysates of C2 toxin-treated cells and identified the FK506-binding protein (FKBP) 51 as a novel interaction partner of C2I in vitro and in intact mammalian cells. Prompted by this finding, we used the specific pharmacological inhibitor FK506 to investigate whether the PPIase activity of FKBPs plays a role during membrane translocation of C2 toxin. Treatment of cells with FK506 protected cultured cells from intoxication with C2 toxin. Moreover, FK506 inhibited the pH-dependent translocation of C2I across membranes into the cytosol but did not interfere with the enzyme activity of C2I or binding of C2 toxin to cells. Furthermore, FK506 treatment delayed intoxication with the related binary actin ADP-ribosylating toxins from Clostridium perfringens (iota toxin) and Clostridium difficile (CDT) but not with the Rho-glucosylating Clostridium difficile toxin A (TcdA). In conclusion, our results support the hypothesis that clostridial binary actin-ADP-ribosylating toxins share a specific FKBP-dependent translocation mechanism during their uptake into mammalian cells.     

Cellular uptake of Clostridium botulinum C2 toxin: membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain

The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited.     

Fate and action of ricin in rat liver in vivo: translocation of endocytosed ricin into cytosol and induction of intrinsic apoptosis by ricin B‐chain

Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome‐associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5–90 min) and was later (15–90 min) partially translocated to cytosol as A‐ and B‐chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell‐free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time‐dependent translocation of ricin across the endosomal membrane occurred in cell‐free endosomes. Endosome‐located thioredoxin reductase‐1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase‐9 and caspase‐3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B‐chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B‐chain with mitochondria is involved in ricin‐induced apoptosis.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Oxidative stress resistance through blocking Hsp60 translocation followed by SAPK/JNK inhibition in aged human diploid fibroblasts

The stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) pathway is a well‐known senescence‐related stress activated protein kinase. Multiple environmental stresses induce programmed cell death, such as apoptosis. Normal human diploid fibroblast (HDF) cells have a limited life span in vitro, halting proliferation after a fixed number of cell divisions. Aged passage HDF showed resistance to oxidative stress involving heat shock proteins (Hsp60) through a mechanism involving the translocation of Hsp60 from the mitochondria to the cytosol. The present study showed that the translocation of Hsp60 from the mitochondria to the cytosol followed by high levels of p‐SAPK/JNK activation as a result of oxidative stress was observed in the young cells only. The inhibition of SAPK/JNK activation by SP600125 under oxidative stress almost completely blocked the translocation of Hsp60 in both young and aged cells. This suggests that aged HDF cells are resistant to oxidative stress by blocking the translocation of Hsp60 from the mitochondria to the cytosol followed by SAPK/JNK inhibition. Overall, the mechanism of resistance by oxidative stress in aged cells is induced by blocked of the translocation of Hsp60 followed by SAPK/JNK inactivation. Copyright © 2008 John Wiley & Sons, Ltd.     

Exploring ligand recognition,selectivity and dynamics of TPR domains of chloroplast Toc64 and mitochondria Om64 from Arabidopsis thaliana

The study aims to gain insight into the mode of ligand recognition by tetratricopeptide repeat (TPR) domains of chloroplast translocon at the outer envelope of chloroplast (Toc64) and mitochondrial Om64, two paralogous proteins that mediate import of proteins into chloroplast and mitochondria, respectively. Chaperone proteins associate with precursor proteins in the cytosol to maintain them in a translocation competent conformation and are recognized by Toc64 and Om64 that are located on the outer membrane of the target organelle. Heat shock proteins (Hsp70) and Hsp90 are two chaperones, which are known to play import roles in protein import. The C‐termini of these chaperones are known to interact with the TPR domain of chloroplast Toc64 and mitochondrial Om64 in Arabidopsis thaliana (At). Using a molecular dynamics approach and binding energy calculations, we identify important residues involved in the interactions. Our findings suggest that the TPR domain from AtToc64 has higher affinity towards C‐terminal residues of Hsp70. The interaction occurs as the terminal helices move towards each other enclosing the cradle on interaction of AtHsp70 with the TPR domain. In contrast, the TPR domain from AtOm64 does not discriminate between the C‐termini of Hsp70 and Hsp90. These binding affinities are discussed with respect to our knowledge of protein targeting and specificity of protein import into endosymbiotic organelles in plant cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Uptake of binary actin ADP-ribosylating toxins

The focus of this article is on the cellular uptake mechanism of the family of binary actin ADP-ribosylating toxins from clostridia. These toxins are special-type AB toxins, because they are composed of two nonlinked proteins, which have to assemble on the surface of eukaryotic cells to act cytotoxically. The enzymatically active component (A), ADP-ribosylates G-actin in the cytosol of target cells. This leads to a complete depolymerization of the actin filaments and, thereby, to rounding up of cultured cells. The second component of these toxins, the binding/translocation component (B), mediates the transport of the enzyme component into the cytosol.     

Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I, which mono-ADP-ribosylates actin in eukaryotic cells. Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol. We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90. Here, we demonstrate that cyclosporin A, which inhibits the peptidyl-prolyl cis / trans isomerase activity of cyclophilins, inhibited intoxication of cells with C2 toxin and prevented uptake of C2I into the cytosol. Cyclosporin A blocked the pH-dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes. In vitro , the addition of cytosol to C2 toxin-loaded endosomes induced translocation of C2I activity into the cytosol, which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A. Pull-down experiments with lysates from C2 toxin-treated cells revealed specific binding of cyclophilin A to the N-terminal domain of C2I. In conclusion, our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of C2 toxin into mammalian cells.     

Selective and specific internalization of clostridial C3 ADP‐ribosyltransferases into macrophages and monocytes

The C3 transferases from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) mono‐ADP‐ribosylate and thereby inactivate RhoA, ‐B and ‐C of eukaryotic cells. Due to their extremely poor cellular uptake, C3 transferases were supposed to be exoenzymes rather than exotoxins, challenging their role in pathogenesis. Here, we report for the first time that low concentrations of both C3lim and C3bot are selectively internalized into macrophages/monocytes in less than 3 h, inducing the reorganization of the actin cytoskeleton by ADP‐ribosylation of Rho. We demonstrate that C3 transferases are internalized into the cytosol of macrophages/monocytes via acidified early endosomes. Bafilomycin A1, an inhibitor of endosomal acidification, protected J774A.1 macrophages and human promyelotic leukaemia cells (HL‐60) from intoxication by C3. Moreover, confocal laser scanning microscopy revealed colocalization of C3 with early endosomes. An extracellular acidic pulse enabled direct translocation of cell surface‐bound C3 across the cytoplasmic membrane to the cytosol. In line with this finding, both C3 proteins exhibited membrane activity in lipid bilayer membranes only under acidic conditions (pH < 5.5). In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis.     

Structure of the ATP‐binding domain of Plasmodium falciparum Hsp90

Hsp90 is an important cellular chaperone and attractive target for therapeutics against both cancer and infectious organisms. The Hsp90 protein from the parasite Plasmodium falciparum, the causative agent of malaria, is critical for this organism's survival; the anti‐Hsp90 drug geldanamycin is toxic to P. falciparum growth. We have solved the structure of the N‐terminal ATP‐binding domain of P. falciparum Hsp90, which contains a principal drug‐binding pocket, in both apo and ADP‐bound states at 2.3 Å resolution. The structure shows that P. falciparum Hsp90 is highly similar to human Hsp90, and likely binds agents such as geldanamycin in an identical manner. Our results should aid in the structural understanding of Hsp90‐drug interactions in P. falciparum, and provide a scaffold for future drug‐discovery efforts. Proteins 2010; © 2010 Wiley‐Liss, Inc.