scholarly journals Structure of a Tc holotoxin pore provides insights into the translocation mechanism

2019 ◽  
Author(s):  
Daniel Roderer ◽  
Oliver Hofnagel ◽  
Roland Benz ◽  
Stefan Raunser
Keyword(s):  
Conformational Transition ◽  
Target Cells ◽  
Enzyme Binding ◽  
Ph Shift ◽  
Head Groups ◽  
Toxin Complex ◽  
Translocation Mechanism

AbstractTc toxins are modular toxin systems that are composed of a pentameric membrane translocator (TcA) and a cocoon (TcB and TcC) encapsulating the toxic enzyme. Binding of Tcs to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. Here, we show in atomic detail an assembled Tc toxin complex fromP. luminescensin the membrane. We find that the five TcA protomers conformationally adapt to fit around the cocoon during prepore-to-pore transition. The architecture of the Tc toxin complex also allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Mammalian lipids with zwitterionic head groups are preferred over other lipids for Tc toxin integration. The translocated toxic enzyme, which can be partially visualized, transiently interacts with alternating negative charges and hydrophobic stretches.

scholarly journals Structure of a Tc holotoxin pore provides insights into the translocation mechanism

2019 ◽  
Vol 116 (46) ◽  
pp. 23083-23090 ◽  
Author(s):  
Daniel Roderer ◽  
Oliver Hofnagel ◽  
Roland Benz ◽  
Stefan Raunser
Keyword(s):  
Pathogenic Bacteria ◽  
Conformational Transition ◽  
Target Cells ◽  
Photorhabdus Luminescens ◽  
C Terminus ◽  
Head Groups ◽  
Toxin Complex ◽  
High Resolution Structure ◽  
Translocation Mechanism ◽  
Resolution Structure

Tc toxins are modular toxin systems of insect and human pathogenic bacteria. They are composed of a 1.4-MDa pentameric membrane translocator (TcA) and a 250-kDa cocoon (TcB and TcC) encapsulating the 30-kDa toxic enzyme (C terminus of TcC). Binding of Tc toxins to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. A high-resolution structure of a holotoxin embedded in membranes is missing, leaving open the question of whether TcB-TcC has an influence on the conformational transition of TcA. Here we show in atomic detail a fully assembled 1.7-MDa Tc holotoxin complex from Photorhabdus luminescens in the membrane. We find that the 5 TcA protomers conformationally adapt to fit around the cocoon during the prepore-to-pore transition. The architecture of the Tc toxin complex allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Importantly, assembly of the holotoxin at the membrane results in spontaneous translocation of the toxic enzyme, indicating that this process is not driven by a proton gradient or other energy source. Mammalian lipids with zwitterionic head groups are preferred over other lipids for the integration of Tc toxins. In a nontoxic Tc toxin variant, we can visualize part of the translocating toxic enzyme, which transiently interacts with alternating negative charges and hydrophobic stretches of the translocation channel, providing insights into the mechanism of action of Tc toxins.

scholarly journals Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1

2002 ◽  
Vol 159 (2) ◽  
pp. 207-216 ◽  
Author(s):  
Billy Tsai ◽  
Tom A. Rapoport
Keyword(s):  
Toxic Effect ◽  
Cholera Toxin ◽  
Disulfide Bond ◽  
Protein Disulfide Isomerase ◽  
Reduced Form ◽  
Target Cells ◽  
Disulfide Isomerase ◽  
Toxin Complex ◽  
Protein Disulfide ◽  
Er Membrane

The toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain. This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol. The polypeptide is initially unfolded by binding to the reduced form of PDI. We show that upon oxidation of the COOH-terminal disulfide bond in PDI by the enzyme Ero1, the A1 chain is released. Both yeast Ero1 and the mammalian Ero1α isoform are active in this reaction. Ero1 has a preference for the PDI–toxin complex. We further show that the complex is transferred to a protein at the lumenal side of the ER membrane, where the unfolded toxin is released from PDI by the action of Ero1. Taken together, our results identify Ero1 as the enzyme mediating the release of unfolded CT from PDI and characterize an additional step in retrotranslocation of the toxin.

scholarly journals Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90

2011 ◽  
Vol 79 (10) ◽  
pp. 3913-3921 ◽  
Author(s):  
Eva Kaiser ◽  
Claudia Kroll ◽  
Katharina Ernst ◽  
Carsten Schwan ◽  
Michel Popoff ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Clostridium Perfringens ◽  
Cyclophilin A ◽  
Target Cells ◽  
Membrane Translocation ◽  
Content Type ◽  
Translocation Mechanism ◽  
Adp Ribosyltransferase ◽  
Endosomal Vesicles

ABSTRACTSome hypervirulent strains ofClostridium difficileproduce the binary actin-ADP-ribosylating toxinC. difficiletransferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity ofC. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin fromClostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin Ain vitro. In combination with our recently obtained data on the C2 toxin fromC. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.

scholarly journals Self-Assembling β-Glucan Nanomedicine for the Delivery of siRNA

Biomedicines ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 497
Author(s):  
Kyungwoo Lee ◽  
Daejin Min ◽  
Yonghyun Choi ◽  
Semi Yoon ◽  
Jaehee Jang ◽  
...  
Keyword(s):  
Drug Delivery Systems ◽  
Conformational Transition ◽  
Genetic Material ◽  
Transition Process ◽  
Target Cells ◽  
Gene Delivery System ◽  
Small Interfering Rnas ◽  
Self Assembling ◽  
High Gene ◽  
Design And Manufacture

We aimed to design and manufacture a transporter capable of delivering small interfering RNAs (siRNAs) into the skin without causing any damage. β-glucans are unique chiral polysaccharides with well-defined immunological properties and supramolecular wrapping ability. However, the chiral properties of these polymers have hardly been applied in drug delivery systems. In this study, β-glucan nanoparticles were designed and manufactured to deliver genetic material to the target cells. The β-glucan molecules were self-assembled with an siRNA into nanoparticles of 300–400 nm in diameter via a conformational transition process, in order to construct a gene delivery system. The assembled gene nanocarriers were associated with high gene-loading ability. The expression and efficiency of siRNA were verified after its delivery via β-glucan. Our results provide evidence that β-glucan nanoparticles can be effectively used to deliver siRNA into the cells.

scholarly journals Truncation of the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Defines Elements Required for Fusion, Incorporation, and Infectivity

2009 ◽  
Vol 83 (22) ◽  
pp. 11588-11598 ◽  
Author(s):  
Ling Yue ◽  
Liang Shang ◽  
Eric Hunter
Keyword(s):  
Amino Acids ◽  
Target Cells ◽  
Amino Acid Residues ◽  
Hydrophobic Residues ◽  
Head Groups ◽  
Immunodeficiency Virus ◽  
Hiv Type 1 ◽  
Membrane Spanning ◽  
Hiv 1

ABSTRACT The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a “snorkeling” model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.

Ultrastructural Changes in Tumor Cells During Human Peripheral Blood Monocyte-Mediated Cytotoxicity

1981 ◽  
Vol 39 ◽  
pp. 452-453
Author(s):  
K. E. Muse ◽  
D. G. Fischer ◽  
H. S. Koren
Keyword(s):  
Target Cell ◽  
Human Peripheral Blood ◽  
Mononuclear Phagocytes ◽  
Ultrastructural Changes ◽  
Target Cells ◽  
Limited Information ◽  
Blood Monocytes ◽  
Tumoricidal Activity ◽  
Activated State

Mononuclear phagocytes, a pluripotential cell line, manifest an array of basic extracellular functions. Among these physiological regulatory functions is the expression of spontaneous cytolytic potential against tumor cell targets.The limited observations on human cells, almost exclusively blood monocytes, initially reported limited or a lack of tumoricidal activity in the absence of antibody. More recently, freshly obtained monocytes have been reported to spontaneously impair the biability of tumor target cells in vitro (Harowitz et al., 1979; Montavani et al., 1979; Hammerstrom, 1979). Although the mechanism by which effector cells express cytotoxicity is poorly understood, discrete steps can be distinguished in the process of cell mediated cytotoxicity: recognition and binding of effector to target cells,a lethal-hit stage, and subsequent lysis of the target cell. Other important parameters in monocyte-mediated cytotoxicity include, activated state of the monocyte, effector cell concentrations, and target cell suseptibility. However, limited information is available with regard to the ultrastructural changes accompanying monocyte-mediated cytotoxicity.

The Effect of Androgen Depletion and Replacement on the Epithelium of the Seminal Vesicle of the Aging Rat

1975 ◽  
Vol 33 ◽  
pp. 590-591
Author(s):  
Venita F. Allison
Keyword(s):  
Cell Proliferation ◽  
Seminal Vesicle ◽  
Male Rats ◽  
Target Cells ◽  
Cell Regeneration ◽  
Secretory Function ◽  
Electron Microscopic ◽  
Aging Rat ◽  
Microscopic Studies ◽  
Androgen Depletion

In 1930, Moore, Hughes and Gallager reported that after castration seminal vesicle epithelial cell atrophy occurred and that cell regeneration could be achieved with daily injections of testis extract. Electron microscopic studies have confirmed those observations and have shown that testosterone injections restore the epithelium of the seminal vesicle in adult castrated male rats. Studies concerned with the metabolism of androgens point out that dihydrotestosterone stimulates cell proliferation and that other metabolites of testosterone probably influence secretory function in certain target cells.Although the influence of androgens on adult seminal vesicle epithelial cytology is well documented, little is known of the effect of androgen depletion and replacement on those cells in aging animals. The present study is concerned with the effect of castration and testosterone injection on the epithelium of the seminal vesicle of aging rats.

Microstructural morphology of sphingolipid dispersions as investigated by freeze-fracture EM

1995 ◽  
Vol 53 ◽  
pp. 944-945
Author(s):  
Vitthal S. Kulkarni ◽  
Wayne H. Anderson ◽  
Rhoderick E. Brown
Keyword(s):  
Acyl Chain ◽  
Biological Significance ◽  
Freeze Fracture ◽  
Fatty Acyl ◽  
Fatty Acyl Moiety ◽  
Aqueous Dispersions ◽  
Head Groups ◽  
Lipid Species ◽  
Microstructural Morphology ◽  
Layer Chromatography

The biological significance of the sphingomyelins (SM) and monoglycosylated sphingolipids like galactosylceramides (GalCer) are well documented Our recent investigation showed tubular bilayers in the aqueous dispersions of N-nervonoyl GalCer [N-(24:lΔ15,cls) GalCer] (a major fatty acyl moiety of natural GalCer). To determine the influence of lipid head groups on the resulting mesophasic morphology, we investigated microstructural self-assemblies of N-nervonoyl-SM [N-(24:1 Δ15,cls) SM; the second most abundant sphingomyelin in mammalian cell membranes], 1- palmitoyl-2-nervonoyl phosphatidylcholine [PNPC] (the lipid species with the same acyl chain configuration as in N-(24: 1) GalCer) and also compared it with egg-SM by freeze-fracture EM.Procedures for synthesizing and purifying N-(24:1) GalCer, N-(24:1) SM, and PNPC have been reported . Egg-SM was purchased from Avanti Polar Lipids, Alabaster AL. All lipids were >99% pure as checked by thin layer chromatography. Lipid dispersions were prepared by hydrating dry lipid with phosphate buffer (pH 6.6) at 80-90°C (3-5 min), vigorously vortexing (1 min) and repeating this procedure for three times prior to three freeze-thaw cycles.

Reduction of Potassium in Kidney Proximal Tubules to Aid in Electron Probe X-Ray Microanalysis of Calcium

1985 ◽  
Vol 43 ◽  
pp. 474-475
Author(s):  
A. LeFurgey ◽  
P. Ingram ◽  
L.J. Mandel
Keyword(s):  
Smooth Muscle ◽  
Proximal Tubule ◽  
Electron Probe ◽  
Clinical Applications ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Target Cells ◽  
X Ray ◽  
Freeze Dried

For quantitative determination of subcellular Ca distribution by electron probe x-ray microanalysis, decreasing (and/or eliminating) the K content of the cell maximizes the ability to accurately separate the overlapping K Kß and Ca Kα peaks in the x-ray spectra. For example, rubidium has been effectively substituted for potassium in smooth muscle cells, thus giving an improvement in calcium measurements. Ouabain, a cardiac glycoside widely used in experimental and clinical applications, inhibits Na-K ATPase at the cell membrane and thus alters the cytoplasmic ion (Na,K) content of target cells. In epithelial cells primarily involved in active transport, such as the proximal tubule of the rabbit kidney, ouabain rapidly (t1/2= 2 mins) causes a decrease2 in intracellular K, but does not change intracellular total or free Ca for up to 30 mins. In the present study we have taken advantage of this effect of ouabain to determine the mitochondrial and cytoplasmic Ca content in freeze-dried cryosections of kidney proximal tubule by electron probe x-ray microanalysis.

The effects of Cyclosporin-A (CYA) on the Ultrastructure of Gingival Mast Cells (GMC) in Behcet's disease (BD)

1990 ◽  
Vol 48 (3) ◽  
pp. 358-359
Author(s):  
Oktay Arda ◽  
Ulkü Noyan ◽  
Selgçk Yilmaz ◽  
Mustafa Taşyürekli ◽  
İsmail Seçkin ◽  
...  
Keyword(s):  
Unknown Etiology ◽  
Control Group ◽  
Target Cells ◽  
Gingival Hyperplasia ◽  
Cellular Activity ◽  
Direct Relation ◽  
Immune Disease ◽  
Genital Ulceration ◽  
Functional Changes ◽  
The Third

Turkish dermatologist, H. Beheet described the disease as recurrent triad of iritis, oral aphthous lesions and genital ulceration. Auto immune disease is the recent focus on the unknown etiology which is still being discussed. Among the other immunosupressive drugs, CyA included in it's treatment newly. One of the important side effects of this drug is gingival hyperplasia which has a direct relation with the presence of teeth and periodontal tissue. We are interested in the ultrastructure of immunocompetent target cells that were affected by CyA in BD.Three groups arranged in each having 5 patients with BD. Control group was the first and didn’t have CyA treatment. Patients who had CyA, but didn’t show gingival hyperplasia assembled the second group. The ones displaying gingival hyperplasia following CyA therapy formed the third group. GMC of control group and their granules are shown in FIG. 1,2,3. GMC of the second group presented initiation of supplementary cellular activity and possible maturing functional changes with the signs of increased number of mitochondria and accumulation of numerous dense cored granules next to few normal ones, FIG. 4,5,6.

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