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- Title
- CELLULAR AND MOLECULAR MECHANISMS OF TOXIN RESISTANCE FOR ENDOPLASMIC RETICULUM TRANSLOCATING TOXINS.
- Creator
-
Massey, Christopher, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
The endoplasmic reticulum (ER) is the site of co- and post-translational modification for secretory proteins. In order to prevent vesicular transport and secretion of misfolded or misassembled proteins, a highly regulated mechanism called ER-associated degradation (ERAD) is employed. This pathway recognizes misfolded proteins in the ER lumen and targets them to the cytosol for ubiquitination and subsequent degradation via the 26S proteasome. Sec61 and Derlin-1 are ER pores through which...
Show moreThe endoplasmic reticulum (ER) is the site of co- and post-translational modification for secretory proteins. In order to prevent vesicular transport and secretion of misfolded or misassembled proteins, a highly regulated mechanism called ER-associated degradation (ERAD) is employed. This pathway recognizes misfolded proteins in the ER lumen and targets them to the cytosol for ubiquitination and subsequent degradation via the 26S proteasome. Sec61 and Derlin-1 are ER pores through which export occurs. AB-type protein toxins such as cholera toxin (CT), Shiga toxin (ST), exotoxin A (ETA), and ricin have evolved means of exploiting the ERAD pathway in order to reach their cytosolic targets. AB-type protein toxins consist of a catalytic A-subunit and a cell-binding B-subunit. The B-subunit recognizes cell surface receptors for the toxin. This begins a series of vesicle trafficking events, collectively termed retrograde trafficking, that lead to the ER. Dissociation of the A and B subunits occurs in the ER, and only the A subunit enters the cytosol. The exact mechanism of A subunit translocation from the ER to the cytosol is unknown. Toxin translocation occurs through a pore in the ER membrane. Exit through the pore requires the toxin to be in an unfolded conformation. The current model for toxin translocation proposes that ER chaperones actively unfold the toxin A chain for translocation. After the translocation event, the toxin spontaneously refolds to an active conformation. Our model suggests that unfolding in the ER is spontaneous and refolding in the cytosol is dependent upon cytosolic chaperones. Based on our model, we hypothesize that blockage of the A subunit unfolding and/or the ERAD translocation step will confer a phenotype of non-harmful multi-toxin resistance to cells. In support of this model, we have shown that, at 37ºC, the isolated catalytic subunit of cholera toxin (CTA1) is in an unfolded and protease sensitive confirmation that identifies the toxin as misfolded by the ERAD pathway. Stabilization of CTA1 via glycerol inhibits the loss of its tertiary structure. This stabilization results in decreased translocation from the ER to the cytosol and increased secretion of CTA1 to the extracellular medium. Treatment with glycerol also prevents CTA1 degradation by the 20S proteasome in vitro. These data indicate that the thermal stability of CTA1 plays an important role in intoxication. These data also suggest that stabilization of CTA1 tertiary structure is a potential target for therapeutic agents. Our model asserts that CTA1 behaves as a normal ERAD substrate upon dissociation from the holotoxin. In support of this model, we have shown that the ER luminal protein HEDJ, known to be involved in ERAD, interacts with CTA1. The interactions between HEDJ and CTA1 occur only at temperatures in which the toxin is in an unfolded conformation. We have also shown that HEDJ does not affect the thermally stability of CTA1 since there is no alteration in its pattern of temperature-dependent protease sensitivity. Alteration of the normal HEDJ-CTA1 interaction via a dominant-negative HEDJ construct resulted in decreased translocation from the ER to the cytosol and, as a result, decreased intoxication. Our work demonstrated toxin resistance can result through effects on toxin structure or ERAD chaperones. To identify other potential inhibitors, we developed a novel assay to detect the activity of other AB toxins and compared it with an established toxicity assay. We generated a Vero cell line that expressed a destabilized variant of enhanced green fluorescent protein (EGFP). These cells were used to monitor the Stx-induced inhibition of protein synthesis by monitoring the loss of EGFP fluorescence from cells. We screened a panel of 13 plant compounds, and indentified grape seed extract and grape pomace extract as inhibitors of Stx activity. Grape seed extract and grape pomace extract were also shown to block the toxic activities of ETA and ricin, providing the basis for a future high-throughput screen for multi-toxin inhibitors.
Show less - Date Issued
- 2009
- Identifier
- CFE0002925, ucf:47999
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002925
- Title
- THE STRUCTURAL AND FOLDING CHARACTERISTICS OF THE PLASMID-ENCODED TOXIN FROM ENTEROAGGREGATIVE ESCHERICHIA COLI.
- Creator
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Scaglione, Patricia, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a member of the autotransporter subfamily termed SPATE (serine protease autotransporters of Enterobacteriaceae). Autotransporters, which are the most common Gram-negative secreted virulence factors, contain three functional domains: an amino terminal leader sequence, a mature protein or passenger domain, and a carboxy-terminal β domain. The leader sequence targets the protein to the periplasmic space and the β...
Show morePlasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a member of the autotransporter subfamily termed SPATE (serine protease autotransporters of Enterobacteriaceae). Autotransporters, which are the most common Gram-negative secreted virulence factors, contain three functional domains: an amino terminal leader sequence, a mature protein or passenger domain, and a carboxy-terminal β domain. The leader sequence targets the protein to the periplasmic space and the β domain then forms a β-barrel pore in the outer membrane of the bacterium which allows the passenger domain to enter the external milieu. In some cases the passenger domain is cleaved from the β-barrel at the extracellular surface to release a soluble toxin. This is thought to be a self-contained process that does not require chaperones or ATP for folding and export of the passenger domain. Pet produces cytotoxic effects through cleavage of its target, the actin-binding protein α- fodrin. Pet is secreted into the extracellular environment, but its target lies within the cytosol. To reach its target, Pet moves from the cell surface to the ER where it triggers ER-associated degradation (ERAD) to enter the cytosol. ERAD is a normal cellular process in which improperly folded proteins are exported from the ER to the cytosol for degradation. Other toxins that utilize this pathway are AB toxins such as cholera toxin (CT) and ricin. The A subunits of these toxins are thermally unstable, and this facilitates their ERAD-dependent translocation into the cytosol. Pet, however, is not an AB toxin. We predict that thermal unfolding is not the mechanism Pet employs to exploit ERAD. It was necessary to purify the toxin first in order to study the structural properties and ER export of Pet. Surprisingly, purified Pet eluted as two close peaks by size exclusion chromatography. Both peaks were Pet as demonstrated through immunoblotting. The folding efficiency of autotransporters has not been extensively elucidated, and based on our purification results, we hypothesized that there is inefficiency in the folding of autotransporters, specifically Pet. A toxicity assay showed that Pet peak one did not display cytopathic activity while Pet peak two did. CD and fluorescence spectroscopy measurements also detected structural differences between the two variants of Pet and demonstrated that Pet peak one was an unfolded variant of Pet peak two. Native gel electrophoresis and biophysical measurements indicated that Pet peak one did not exist as a dimer or aggregate. Our results indicate there are two forms of Pet, and thus the folding process of autotransporters appears to be inherently inefficient. Active Pet (peak two) was used for further biophysical measurements and biochemical assays. Circular dichroism and fluorescence spectroscopy showed that the secondary and tertiary structures of Pet are maintained at physiological temperature, 37°C. Thermal unfolding of Pet occurred at temperatures above 50°C. Fluorescence quenching of Pet was also performed and demonstrated that, at 37°C, there are solvent-exposed aromatic amino acids. The slight structural alterations to Pet at physiological temperature as well as the exposed hydrophobic residues could trigger ERAD. In addition, a modeled structure of Pet revealed a hydrophobic loop which is surface-exposed and a likely target for toxin-ERAD interactions. The data suggests that translocation of Pet mediated by ERAD can occur by a mechanism different from certain AB toxins. An open, hydrophobic conformation likely triggers ERAD, but may also contribute to poor folding.
Show less - Date Issued
- 2008
- Identifier
- CFE0002399, ucf:47761
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002399
