scholarly journals Proton-coupled protein transport through the anthrax toxin channel

2008 ◽  
Vol 364 (1514) ◽  
pp. 209-215 ◽  
Author(s):  
Alan Finkelstein
Keyword(s):  
Protein Transport ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Low Ph ◽  
Phospholipid Bilayers ◽  
Anthrax Toxin ◽  
Large Hole ◽  
The Third ◽  
Folded Proteins ◽  
Acidic Vesicle

Anthrax toxin consists of three proteins (approx. 90 kDa each): lethal factor (LF); oedema factor (OF); and protective antigen (PA). The former two are enzymes that act when they reach the cytosol of a targeted cell. To enter the cytosol, however, which they do after being endocytosed into an acidic vesicle compartment, they require the third component, PA. PA (or rather its proteolytically generated fragment PA 63 ) forms at low pH a heptameric β-barrel channel, (PA 63 ) 7 , through which LF and OF are transported—a phenomenon we have demonstrated in planar phospholipid bilayers. It might appear that (PA 63 ) 7 simply forms a large hole through which LF and OF diffuse. However, LF and OF are folded proteins, much too large to fit through the approximately 15 Å diameter (PA 63 ) 7 β-barrel. This paper discusses how the (PA 63 ) 7 channel both participates in the unfolding of LF and OF and functions in their translocation as a proton–protein symporter.

scholarly journals Anthrax Toxin Characterization

2002 ◽  
Vol 45 (1) ◽  
pp. 3-5 ◽  
Author(s):  
Jiří Patočka ◽  
Miroslav Špliňo
Keyword(s):  
Blood Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
White Blood Cells ◽  
The Body ◽  
Anthrax Toxin ◽  
Signaling System ◽  
Animal Cells ◽  
The Third ◽  
Edema Factor

The anthrax toxin comprises three proteins. When they work together, they can kill humans, especially after spores of the bacteria have been inhaled. One anthrax protein, called protective antigen (PA), chaperones the two other toxins into human or animal cells and shields them from the body’s immune system. The second, lethal factor (LF), destroys the white blood cells that hosts send in defence. The third toxin molecule, edema factor (EF), hijacks the signaling system in the body. This disrupts the energy balance of cells and leads to them accumulating fluid and complete destroy of cells.

scholarly journals A kinetic analysis of protein transport through the anthrax toxin channel

2011 ◽  
Vol 137 (6) ◽  
pp. 521-531 ◽  
Author(s):  
Daniel Basilio ◽  
Paul K. Kienker ◽  
Stephen W. Briggs ◽  
Alan Finkelstein
Keyword(s):  
Protein Transport ◽  
Protective Antigen ◽  
Single Channel ◽  
Protein Translocation ◽  
Lethal Factor ◽  
Phospholipid Bilayer ◽  
Anthrax Toxin ◽  
Cumulative Distribution ◽  
Endosomal Membrane ◽  
Edema Factor

Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell’s endosomal membrane, disrupting cellular homeostasis. (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LFN, the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LFN molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LFNs. In this paper, we show both in macroscopic and single-channel experiments that even with only one LFN bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LFN bound. We also present a simple electrodiffusion model of translocation in which LFN is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

scholarly journals The chymotrypsin-sensitive site, FFD315, in anthrax toxin protective antigen is required for translocation of lethal factor.

1994 ◽  
Vol 269 (46) ◽  
pp. 29039-29046
Author(s):  
Y Singh ◽  
K R Klimpel ◽  
N Arora ◽  
M Sharma ◽  
S H Leppla
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Sensitive Site

scholarly journals Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

2006 ◽  
Vol 13 (6) ◽  
pp. 671-677 ◽  
Author(s):  
Robert Mabry ◽  
Kathleen Brasky ◽  
Robert Geiger ◽  
Ricardo Carrion ◽  
Gene B. Hubbard ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Systemic Circulation ◽  
Rapid Identification ◽  
Anthrax Toxin ◽  
Soluble Form ◽  
Before And After

ABSTRACT Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.

Oligomerization of Anthrax Toxin Protective Antigen and Binding of Lethal Factor during Endocytic Uptake into Mammalian Cells

1999 ◽  
Vol 67 (4) ◽  
pp. 1853-1859
Author(s):  
Yogendra Singh ◽  
Kurt R. Klimpel ◽  
Seema Goel ◽  
Prabodha K. Swain ◽  
Stephen H. Leppla
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin

Anthrax toxin complexes: heptameric protective antigen can bind lethal factor and edema factor simultaneously

2004 ◽  
Vol 322 (1) ◽  
pp. 258-262 ◽  
Author(s):  
Ruth-Anne L. Pimental ◽  
Kenneth A. Christensen ◽  
Bryan A. Krantz ◽  
R. John Collier
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Edema Factor

scholarly journals Cytosolic Delivery and Characterization of the TcdB Glucosylating Domain by Using a Heterologous Protein Fusion

2001 ◽  
Vol 69 (1) ◽  
pp. 599-601 ◽  
Author(s):  
Lea M. Spyres ◽  
Maen Qa'Dan ◽  
Amy Meader ◽  
James J. Tomasek ◽  
Eric W. Howard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Small Gtpases ◽  
Anthrax Toxin ◽  
Coding Region ◽  
Protein Fusion ◽  
Actin Reorganization ◽  
Amino Terminal ◽  
Cell Extracts

ABSTRACT TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB1-556 coding region were constructed, expressed, and purified from Escherichia coli. LFnTcdB1-556was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB1-556 plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.

scholarly journals Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins

2011 ◽  
Vol 137 (4) ◽  
pp. 343-356 ◽  
Author(s):  
Daniel Basilio ◽  
Laura D. Jennings-Antipov ◽  
Karen S. Jakes ◽  
Alan Finkelstein
Keyword(s):  
Secondary Structure ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Bilayer Membranes ◽  
Endosomal Membrane ◽  
Edema Factor ◽  
N Terminus ◽  
Minimum Number ◽  
Pass Through

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA63)7, in the host cell’s endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA63)7 incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ∼100 Å–long, 15 Å–wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LFN (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LFN adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LFN in the normal direction but also can, at least partially, translocate LFN in the opposite direction.

scholarly journals Ion selectivity of the anthrax toxin channel and its effect on protein translocation

2015 ◽  
Vol 146 (2) ◽  
pp. 183-192 ◽  
Author(s):  
Aviva Schiffmiller ◽  
Damon Anderson ◽  
Alan Finkelstein
Keyword(s):  
Protective Antigen ◽  
Protein Translocation ◽  
Ion Selectivity ◽  
Lethal Factor ◽  
Phospholipid Bilayer ◽  
Anthrax Toxin ◽  
Terminal Portion ◽  
Bilayer Membranes ◽  
Cation Selectivity ◽  
Edema Factor

Anthrax toxin consists of three ∼85-kD proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). PA63 (the 63-kD, C-terminal portion of PA) forms heptameric channels ((PA63)7) in planar phospholipid bilayer membranes that enable the translocation of LF and EF across the membrane. These mushroom-shaped channels consist of a globular cap domain and a 14-stranded β-barrel stem domain, with six anionic residues lining the interior of the stem to form rings of negative charges. (PA63)7 channels are highly cation selective, and, here, we investigate the effects on both cation selectivity and protein translocation of mutating each of these anionic residues to a serine. We find that although some of these mutations reduce cation selectivity, selectivity alone does not directly predict the rate of protein translocation; local changes in electrostatic forces must be considered as well.

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