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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
- Title
- STUDIES OF NORSPERMIDINE UPTAKE IN DROSOPHILA SUGGEST THE EXISTENCE OF MULTIPLE POLYAMINE TRANSPORT PATHWAYS.
- Creator
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Dieffenbach, Michael, Von Kalm, Laurence, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Polyamines are a class of essential nutrients involved in many basic cellular processes such as gene expression, cell proliferation, and apoptosis. Without polyamines, cell growth is delayed or halted. Cancerous cells require an abundance of polyamines through a combination of synthesis and transport from the extracellular environment. An FDA-approved drug, D,L-?-difluoromethylornithine (DFMO), blocks polyamine synthesis but is ineffective at inhibiting cell growth due to polyamine transport....
Show morePolyamines are a class of essential nutrients involved in many basic cellular processes such as gene expression, cell proliferation, and apoptosis. Without polyamines, cell growth is delayed or halted. Cancerous cells require an abundance of polyamines through a combination of synthesis and transport from the extracellular environment. An FDA-approved drug, D,L-?-difluoromethylornithine (DFMO), blocks polyamine synthesis but is ineffective at inhibiting cell growth due to polyamine transport. Thus, there is a need to develop drugs that inhibit polyamine transport to use in combination with DFMO. Surprisingly, little is known about the polyamine transport system in humans and other eukaryotes. Understanding the transport system would allow us to identify compounds that inhibit polyamine transport, which could then be used in tandem with DFMO to treat cancer. Our laboratory has identified one gene in Drosophila, called CG32000, as a component of this transport system, and numerous other candidate genes remain to be tested. To better characterize this system, this project investigated the ability of the Drosophila transport system to take up a toxic polyamine analogue called norspermidine, with the initial goal of developing a new screening method to find polyamine transport genes. My experiments have demonstrated significant differences in norspermidine uptake and toxicity between C. elegans and Drosophila which may imply a secondary polyamine transport system in higher eukaryotes. In the long term, it is hoped that this thesis will facilitate the development of more effective cancer medications by providing new information about the polyamine transport system.
Show less - Date Issued
- 2018
- Identifier
- CFH2000294, ucf:45869
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000294
- Title
- Structure of reflectin protein probed by solid-state nuclear magnetic resonance.
- Creator
-
Boykin, Tommy, Chen, Bo, Schulte, Alfons, Tatulian, Suren, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
An unusual protein found in squids, termed reflectin, possesses the unique ability for optical reflectivity and proton conductivity. Reflectin has the potential to become optically and electronically tunable, however, the molecular structure of reflectin has yet to be determined. Previous literature has considered using reflectin proteins as diffraction gratings, protonic transistors, and substrates for neural stem cell growth. Applying recombinant protein techniques, we purified the...
Show moreAn unusual protein found in squids, termed reflectin, possesses the unique ability for optical reflectivity and proton conductivity. Reflectin has the potential to become optically and electronically tunable, however, the molecular structure of reflectin has yet to be determined. Previous literature has considered using reflectin proteins as diffraction gratings, protonic transistors, and substrates for neural stem cell growth. Applying recombinant protein techniques, we purified the reflectin 4Ax4 protein. We determined the leucine, tryptophan, and threonine amino acids are in an ordered state by solid-state nuclear magnetic resonance (NMR). Knowing these ordered amino acids is possibly the key to understanding reflectin's natural optical and electrical properties. Understanding the link between reflectin's structure and electrical properties is essential to make the next generation of bioelectronic materials based on inexpensive, natural resources.
Show less - Date Issued
- 2019
- Identifier
- CFE0007433, ucf:52712
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007433
- Title
- Exacerbation of efp- sickness in Escherichia coli by an uncharacterized RNA helicase.
- Creator
-
Wingo, Robert, Moore, Sean, Roy, Herve, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
In Escherichia coli, growth is rate-limited by translation capacity. Stalled ribosomes have profound effects on a cell such as altered mRNA abundance, decreased ribosome availability, and an imbalanced proteome. The absence of elongation factor P (EF-P), a universally conserved transpeptidation enhancer, presents an extreme example of this scenario, wherein ribosomes accumulate disproportionately onto messages that are more slowly translated and cell growth becomes notably impaired. We...
Show moreIn Escherichia coli, growth is rate-limited by translation capacity. Stalled ribosomes have profound effects on a cell such as altered mRNA abundance, decreased ribosome availability, and an imbalanced proteome. The absence of elongation factor P (EF-P), a universally conserved transpeptidation enhancer, presents an extreme example of this scenario, wherein ribosomes accumulate disproportionately onto messages that are more slowly translated and cell growth becomes notably impaired. We discovered that faster-growing cells arise spontaneously in ?efp cultures, suggesting that translation defects could be circumvented by mutating other genes. This thesis presents a genetic and biochemical analysis of a mechanism ?efp cells employ to overcome translation stress. Using a dual luciferase reporter system, we found that transpeptidation remained hindered in the faster growing ?efp cells. Whole genome sequencing of several fast-growing strains revealed mutations in a poorly characterized RNA helicase called HrpA. We determined that deletion of hrpA, or mutations at several conserved residues critical for HrpA's function, was sufficient to improve the fitness of ?efp cells. HrpA is a DEAH-box RNA helicase and represents a large class of enigmatic proteins that use ATP to restructure cellular RNAs; however, it's direct function in cellular physiology has yet to be clearly demonstrated. Several HrpA mutants were engineered to interrogate the molecular mechanism of HrpA and how its function impairs ?efp cells. Complementation in ?efp ?hrpA cells showed that a number of these mutants were unable to restore sickness, suggesting they were defective in key aspects of RNA processing. It was discovered that wild-type HrpA is associated with actively translating ribosomes and several of the inactive HrpA mutants impose substantial deleterious effects on translation and ribosome production. In sum, the work presented here describes a mechanism by which cells overcome translation stress involving a novel genetic and biochemical relationship between EF-P and HrpA.
Show less - Date Issued
- 2018
- Identifier
- CFE0007267, ucf:52178
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007267
- Title
- ATP-Induced Disassembly of CDTB/CDTC Heterodimer of Cytolethal Distending Toxin.
- Creator
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Huhn, George, Teter, Kenneth, Cole, Alexander, Jewett, Mollie, University of Central Florida
- Abstract / Description
-
Cytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi. This fastidious pathogen is the causative agent of genital cancroid. CDT is a heterotrimeric toxin with an AB2 structure consisting of a cell-binding (")B(") domain (CdtA + CdtC) and a catalytic (")A(") domain (CdtB) that has DNase activity. This toxin assembles in the bacterial periplasm that lacks ATP and is secreted into the extracellular environment. After cell...
Show moreCytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi. This fastidious pathogen is the causative agent of genital cancroid. CDT is a heterotrimeric toxin with an AB2 structure consisting of a cell-binding (")B(") domain (CdtA + CdtC) and a catalytic (")A(") domain (CdtB) that has DNase activity. This toxin assembles in the bacterial periplasm that lacks ATP and is secreted into the extracellular environment. After cell binding, CDT is internalized by endocytosis and travels through the endosomes and Golgi before arriving in the endoplasmic reticulum (ER). CdtA is lost from the holotoxin before reaching the Golgi, and CdtB separates from CdtC in the ER. CdtB is then transported into the nucleus, inducing cell cycle arrest and apoptosis. Using disassembly of the AB5 pertussis toxin as a model, we explore that ATP, which is present in the ER lumen but not in the endosomes or Golgi, will cause dissociation of the CdtB/CdtC heterodimer. We have cloned and purified the three individual subunits of the H. ducreyi CDT. When combined, the subunits form a lethal holotoxin. Examining the individual toxin subunits, only CdtB binds with ATP but does not function as an ATPase. CdtB's binding to ATP also does not cause global changes to its secondary structure. After isolating the CdtB/CdtC heterodimer, we have shown the addition of ATP causes CdtC to dissociate from CdtB. The work presented in this Thesis provides a molecular basis for why the CdtB/CdtC heterodimer disassembles after reaching the ER and confirms the novel two-stage disassembly mechanism for CDT, a first in the AB toxin field.
Show less - Date Issued
- 2019
- Identifier
- CFE0007657, ucf:52488
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007657
- Title
- Development of a Non-Human Primate Model for Staphylococcus aureus Nasal Carriage.
- Creator
-
Lasseter, Amanda, Cole, Amy, Naser, Saleh, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Staphylococcus aureus nasal carriage (SANC) is largely asymptomatic, but presents a risk of autoinfection and dissemination to new immunocompromised hosts. SA disease states range from mild cutaneous infections to life-threatening bacteremia. Historically utilized rodent models do not naturally carry SA in the nose, are insufficient in longitudinal SANC experimentation, and lack immune factors that are vital in human clearance of SA. The nasal passages of non-human primates are similar...
Show moreStaphylococcus aureus nasal carriage (SANC) is largely asymptomatic, but presents a risk of autoinfection and dissemination to new immunocompromised hosts. SA disease states range from mild cutaneous infections to life-threatening bacteremia. Historically utilized rodent models do not naturally carry SA in the nose, are insufficient in longitudinal SANC experimentation, and lack immune factors that are vital in human clearance of SA. The nasal passages of non-human primates are similar anatomically and histologically, and reproductive mucosal studies have shown similar immune responses to pathogens and human-relevant microbial profiles. Seventeen captive pigtailed macaques (Macaca nemestrina) were found to naturally carry SA in the nose and pharynx, while topical mupirocin ointment effectively decolonized SA, similar to humans. Colonization was established with a human-relevant inoculum of 104 SA CFUs per nostril in four independent experiments, including with a human isolate (ST398). Autologous and non-autologous macaque strains were carried similarly in load and duration, each surviving over 40 days. Animals that cleared SA showed a rapid neutrophilic innate response, with up-regulation of IL-8, MCP-1, and IL-1? following inoculation, as observed in human hosts. Assessment of the nasal microbiome of pigtailed macaques and humans demonstrated similar relative abundance of the most prevalent genera: Staphylococcus, Corynebacterium, and Acinetobacter. Collectively, these multidimensional analyses provide evidence that the pigtailed macaque is a novel physiological model of human SANC that may be useful for testing novel SA decolonization strategies.
Show less - Date Issued
- 2018
- Identifier
- CFE0007216, ucf:52236
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007216
- Title
- Modeling Disease Impact of Vibrio-Phage Interactions.
- Creator
-
Botelho, Christopher, Shuai, Zhisheng, Nevai, A, Zhang, Teng, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Since the work of John Snow, scientists and medical professionals have understood that individuals develop cholera by means of consuming contaminated water. Despite the knowledge(&)nbsp;of cholera's route of infection, many countries have experienced and still experience endemic cholera. Cholera is caused by the Vibrio cholerae (V. cholerae) bacterium and presents with acute diarrhea and vomiting. If untreated, infected individuals may die due to dehydration. Cholera is a disease that most...
Show moreSince the work of John Snow, scientists and medical professionals have understood that individuals develop cholera by means of consuming contaminated water. Despite the knowledge(&)nbsp;of cholera's route of infection, many countries have experienced and still experience endemic cholera. Cholera is caused by the Vibrio cholerae (V. cholerae) bacterium and presents with acute diarrhea and vomiting. If untreated, infected individuals may die due to dehydration. Cholera is a disease that most commonly affects countries with poor infrastructure and water sanitation. Despite efforts to control cholera in such countries, the disease persists. One such example is Haiti which has been experiencing a cholera outbreak since 2010. While there has been much research in the field of microbiology to understand V. cholerae, there has been comparably less research in the field of mathematical biology to understand the dynamics of V. cholerae in the environment. A mathematical model of V. cholerae incorporating a phage population is coupled with a SIRS disease model to examine the impact of vibrio and phage interaction. It is shown that there might exist two endemic equilibria, besides the disease free equilibrium: one in which phage persist in the environment and one in which the phage fail to persist. Existence and stability of these equilibria are established. Disease control strategies based on vibrio and phage interactions are discussed.
Show less - Date Issued
- 2019
- Identifier
- CFE0007604, ucf:52544
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007604
- Title
- AB Toxins: Recovery from Intoxication and Relative Potencies.
- Creator
-
Cherubin, Patrick, Teter, Kenneth, Naser, Saleh, Jewett, Travis, Zervos, Antonis, University of Central Florida
- Abstract / Description
-
AB-type protein toxins have a catalytic A subunit attached to a cell-binding B subunit. Ricin, Shiga toxin (Stx), exotoxin A, and diphtheria toxin are AB toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. Our overall goal is to help elucidate the cellular basis of intoxication for therapeutic development. According to the current model of intoxication, the effect of AB toxins is irreversible. To test this model, we...
Show moreAB-type protein toxins have a catalytic A subunit attached to a cell-binding B subunit. Ricin, Shiga toxin (Stx), exotoxin A, and diphtheria toxin are AB toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. Our overall goal is to help elucidate the cellular basis of intoxication for therapeutic development. According to the current model of intoxication, the effect of AB toxins is irreversible. To test this model, we developed a system that uses flow cytometry and a fluorescent reporter to examine the cellular potency of toxins that inhibit protein synthesis. Our data show that cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin exposure. This work challenges the prevailing model of intoxication by suggesting ongoing toxin delivery to the cytosol is required to maintain the inhibition of protein synthesis and ultimately cause apoptosis. We also used our system to examine the basis for the greater cellular potency of Stx1 in comparison to Stx2. We found that cells intoxicated with Stx1a behave differently than those intoxicated with Stx2: cells exposed to Stx1a exhibited a population-wide loss of protein synthesis, while cells exposed to Stx2a or Stx2c exhibited a dose-dependent bimodal response in which one subpopulation of cells was unaffected (i.e., no loss of protein synthesis). Additional experiments indicated the identity of the Stx B subunit is a major factor in determining the uniform vs. bimodal response to Stx subtypes. This work provides evidence explaining, in part, the differential toxicity between Stx1 and Stx2. Overall, our collective observations provide experimental support for the development of inhibitors and post-exposure therapeutics that restrict, but not necessarily block, toxin delivery to the host cell.
Show less - Date Issued
- 2019
- Identifier
- CFE0007613, ucf:52523
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007613
- Title
- It takes two to tango: the toxin-chaperone relationship.
- Creator
-
Kellner, Alisha, Teter, Kenneth, Moore, Sean, Cole, Alexander, Harper, James, University of Central Florida
- Abstract / Description
-
Cholera toxin (CT) enters the cell via receptor-mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit (CTA1) is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP-ribosylation of its G-protein target. We have shown that the cytosolic chaperones Hsp90 and Hsc70 are required for CTA1 translocation to the cytosol. We have also shown that both are...
Show moreCholera toxin (CT) enters the cell via receptor-mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit (CTA1) is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP-ribosylation of its G-protein target. We have shown that the cytosolic chaperones Hsp90 and Hsc70 are required for CTA1 translocation to the cytosol. We have also shown that both are able to independently bind and refold CTA1. Using libraries of CTA1-derived peptides, we have identified a single Hsc70 binding site, YYIYVI (CTA1 83-88), within the 192 amino acid protein, as well as two distinct Hsp90 binding sites: an N-terminal RPPDEI (CTA111-16) motif and a C-terminal LDIAPA (CTA1 153-158) motif. The LDIAPA motif is unique to CTA1, but an RPPDEI-like motif is present in four other ER-translocating ADP-ribosylating toxins: pertussis toxin, Pseudomonas aeruginosa exotoxin A, Escherichia coli heat-labile toxin, and Salmonella typhimurium ADP-ribosylating toxin. Using site-directed mutagenesis to further investigate the RPPDEI motif, we found that a modification of either proline residue blocks CTA1 translocation to the cytosol. Our work has identified, for the first time, specific amino acid sequences that are recognized by Hsp90/Hsc70 and are essential for toxin translocation from the ER to the cytosol. CT does not require prolyl isomerases for cellular activity, as is the case for Hsp90-dependent endosome-translocating toxins. We therefore hypothesize that the one or both of the prolines within the RPPDEI motif of CTA1 undergo an isomerization event as CTA1 unfolds in the ER. Furthermore, we predict that the trans- to cis- conformational change of proline(s) is the molecular determinate for the atypical Hsp90 interaction observed with CTA1 and related toxins. Additionally, we have identified Hsp90 and other host factors required for the translocation of pertussis toxin.
Show less - Date Issued
- 2019
- Identifier
- CFE0007661, ucf:52500
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007661
- Title
- Unraveling PDI and its Interaction with AB Toxins.
- Creator
-
Guyette, Jessica, Teter, Kenneth, Self, William, Jewett, Travis, Tatulian, Suren, University of Central Florida
- Abstract / Description
-
Protein disulfide isomerase (PDI) is an essential endoplasmic reticulum (ER) protein that acts as both an oxidoreductase and chaperone. It exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin (Ctx), which is a unique property of PDI. This unfolding allows PDI to disassemble the Ctx holotoxin, which is required for Ctx activity. Here, we investigated the unfolding and refolding property of PDI and how this affects its...
Show moreProtein disulfide isomerase (PDI) is an essential endoplasmic reticulum (ER) protein that acts as both an oxidoreductase and chaperone. It exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin (Ctx), which is a unique property of PDI. This unfolding allows PDI to disassemble the Ctx holotoxin, which is required for Ctx activity. Here, we investigated the unfolding and refolding property of PDI and how this affects its interaction with bacterial toxins. PDI showed remarkable redox-linked conformational resilience that allows it to refold after being thermally stressed. Deletion constructs of PDI showed that both active domains play opposing roles in stability, and can both refold from an unfolded state, indicating that either domain could unfold during its interaction with Ctx. Its ability to refold suggests that the cycle of unfolding and refolding with Ctx is a normal mechanism that prevents protein aggregation. Disruption of this cycle with the polyphenol, quercetin-3-rutinoside, prevented the disassembly of Ctx, which blocked Ctx intoxication of cultured cells. Loss of PDI function was also found to inhibit intoxication with Escherichia coli heat-labile toxin but not with ricin and Shiga toxins. Toxin structure also contributes to efficiency of PDI binding and disassembly, which may explain the differential potencies between toxins. While Ctx and Ltx share similar structures, Ctx is more potent and efficiently disassembled compared to Ltx. We believe that PDI-mediated disassembly is the rate-limiting step in intoxication, thus dictating toxin potency. Overall, PDI can be targeted for a potential therapeutic for many bacterial toxins because of its unique unfolding properties and its key role in cell intoxication.
Show less - Date Issued
- 2019
- Identifier
- CFE0007646, ucf:52511
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007646
- Title
- Mechanisms of Alpha-Synuclein-Induced Neurodegenertaion in Parkinson's Disease and Stroke.
- Creator
-
Belal, Cherine, Chan, Sic, Ebert, Steven, Self, William, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Parkinson's disease (PD) is a debilitating neurodegenerative disorder affecting one million Americans. Despite its social and economic impact, the pathological cascades that lead to neuron dysfunction and degeneration in PD are poorly understood. Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases including PD. The ER is an organelle central to protein folding and intracellular Ca2+ homeostasis. Perturbations of these...
Show moreParkinson's disease (PD) is a debilitating neurodegenerative disorder affecting one million Americans. Despite its social and economic impact, the pathological cascades that lead to neuron dysfunction and degeneration in PD are poorly understood. Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases including PD. The ER is an organelle central to protein folding and intracellular Ca2+ homeostasis. Perturbations of these functions result in ER stress and upregulation of ER stress proteins, of which some have been implicated in counteracting ER stress-induced cell death. The mechanisms that lead to ER stress and how ER stress proteins contribute to the degenerative cascades remain unclear but their understanding is critical to devising effective therapies for PD. Both the accumulation of mutant a-synuclein (aSyn), which causes an inherited form of PD, and the inhibition of mitochondrial complex I function by PD-inducing neurotoxin lead to ER stress. The critical involvement of ER stress in experimental models of PD supports its potential relevance to PD pathogenesis and led us to test the hypothesis whether the homocysteine-inducible ER protein (Herp), an ubiquitin-like domain (UBD) containing ER-resident protein, can counteract mutant Alpha Syn- and neurotoxin- induced pathological cascades.
Show less - Date Issued
- 2011
- Identifier
- CFE0004470, ucf:49310
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004470
- Title
- Signals Delivered By Interleukin-7 Regulate The Activities Of Bim And JunD In T Lymphocytes.
- Creator
-
Ruppert, Shannon, Khaled, Annette, Self, William, Zervos, Antonis, Teter, Kenneth, University of Central Florida
- Abstract / Description
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Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting proliferation and survival. While the survival and proliferative functions of IL-7 are well established, the identities of IL-7 signaling components in pathways other than JAK/STAT, that accomplish these tasks remain poorly defined. To this end, we used IL-7 dependent T-cells to examine those components necessary for cell growth and survival. Our studies revealed two novel signal...
Show moreInterleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting proliferation and survival. While the survival and proliferative functions of IL-7 are well established, the identities of IL-7 signaling components in pathways other than JAK/STAT, that accomplish these tasks remain poorly defined. To this end, we used IL-7 dependent T-cells to examine those components necessary for cell growth and survival. Our studies revealed two novel signal transducers of the IL-7 growth signal: BimL and JunD. IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. After a bioinformatics survey to reveal possible JunD-regulated genes activated early in the IL-7 signaling cascade, our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism, including Pim-1. Pim-1, an IL-7 induced protein, was inhibited upon JNK or JunD inhibition. Our hypothesis that JunD positively regulated proliferation was confirmed when the proliferation of primary CD8+ T-cells cultured with IL-7 was impaired upon treatment with JunD siRNA. These results show that the IL-7 signal is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth through the expression of metabolic factors like HXKII and Pim-1. When metabolic activities are inhibited, cells undergo autophagy, or cell scavenging, to provide essential nutrients. Pro-apoptotic Bim was evaluated for its involvement in autophagy. Bim is a BH3-only member of the Bcl-2 family that contributes to T-cell death. Partial rescue of T-cells occurs when Bim and the interleukin-7 receptor are deleted, implicating Bim in IL-7-deprived T-cell apoptosis. Alternative splicing results in three different isoforms: BimEL, BimL, and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on IL-7 for growth, were used. Loss of Bim in IL-7-deprived T-cells delayed apoptosis, but blocked the degradative phase of autophagy. The conversion of LC3-I to LC3-II was observed in Bim-deficient T-cells, but p62, which is degraded in autolysosomes, accumulated. To explain this, BimL, was found to support acidification of lysosomes associated with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon IL-7 withdrawal only in Bim-containing T-cells, indicating that in these cells autophagy was protective. IL-7 dependent T-cells lacking Bim were insensitive to inhibition of autophagy or lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosome-tracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, associated with intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of functional autolysosomes during autophagy.
Show less - Date Issued
- 2012
- Identifier
- CFE0004435, ucf:49331
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004435
- Title
- Cholera toxin activates the unfolded protein response through an adenylate cyclase-independent mechanism.
- Creator
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Vanbennekom, Neyda, Teter, Kenneth, Tatulian, Suren, Jewett, Mollie, Zervos, Antonis, University of Central Florida
- Abstract / Description
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Cholera toxin (CT) is a bacterial protein toxin responsible for the gastrointestinal disease known as cholera. CT stimulates its own entry into intestinal cells after binding to cell surface receptors. Once internalized, CT is delivered via vesicle-mediated transport to the endoplasmic reticulum (ER), where the CTA1 subunit dissociates from the rest of the toxin and is exported (or translocated) into the cytosol. CTA1 translocates from the ER lumen into the host cytosol by exploiting a host...
Show moreCholera toxin (CT) is a bacterial protein toxin responsible for the gastrointestinal disease known as cholera. CT stimulates its own entry into intestinal cells after binding to cell surface receptors. Once internalized, CT is delivered via vesicle-mediated transport to the endoplasmic reticulum (ER), where the CTA1 subunit dissociates from the rest of the toxin and is exported (or translocated) into the cytosol. CTA1 translocates from the ER lumen into the host cytosol by exploiting a host quality control mechanism called ER-associated degradation (ERAD) that facilitates the translocation of misfolded proteins into the cytosol for degradation. Cytosolic CTA1, however, escapes this fate and is then free to activate its target, heterotrimeric G-protein subunit alpha (Gs?), leading to adenlyate cyclase (AC) hyperactivation and increased cAMP concentrations. This causes the secretion of chloride ions and water into the intestinal lumen. The result is severe diarrhea and dehydration which are the major symptoms of cholera. CTA1's ability to exploit vesicle-mediated transport and ERAD for cytosolic entry demonstrates a potential link between cholera intoxication and a separate quality control mechanism called the unfolded protein response (UPR), which up-regulates vesicle-mediated transport and ERAD during ER stress. Other toxins in the same family such as ricin and Shiga toxin were shown to regulate the UPR, resulting in enhanced intoxication.Here, we show UPR activation by CT, which coincides with a marked increase in cytosolic CTA1 after 4 hours of toxin exposure. Drug induced-UPR activation also increases CTA1 delivery to the cytosol and increases cAMP concentrations during intoxication. We investigated whether CT stimulated UPR activation through Gs? or AC. Chemical activation of Gs? induced the UPR and increased CTA1 delivery to the cytosol. However, AC activation did not increase cytosolic CTA1 nor did it activate the UPR. These data provide further insight into the molecular mechanisms that cause cholera intoxication and suggest a novel role for Gs? during intoxication, which is UPR activation via an AC-independent mechanism.
Show less - Date Issued
- 2013
- Identifier
- CFE0004951, ucf:49560
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004951
- Title
- Cathepsin B Regulates VLDL Secretion Through LFABP Cleavage.
- Creator
-
Thibeaux, Simeon, Siddiqi, Shadab, Kim, Yoon-Seong, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
The liver is tasked with managing the concentration of various metabolites in the blood, and of particular importance is the uptake of free fatty-acid (FFA), as elevated concentrations of FFA are toxic to cells. FFAs are transported across the cell membrane by CD36 and distributed by LFABP to the endoplasmic reticulum (ER), where they are esterified to glycerol, yielding more chemically inert triglyceride (TAG), which is essential to the process of VLDL assembly. VLDL secretion distributes...
Show moreThe liver is tasked with managing the concentration of various metabolites in the blood, and of particular importance is the uptake of free fatty-acid (FFA), as elevated concentrations of FFA are toxic to cells. FFAs are transported across the cell membrane by CD36 and distributed by LFABP to the endoplasmic reticulum (ER), where they are esterified to glycerol, yielding more chemically inert triglyceride (TAG), which is essential to the process of VLDL assembly. VLDL secretion distributes energy rich TAG to peripheral tissues, and its dysfunction leads to hepatic steatosis, which may progress into hepatocellular carcinoma. The present study examined the role of cathepsin B (CatB) in regulating very-low density lipoprotein (VLDL) secretion through liver fatty-acid binding protein (LFABP) cleavage as well as CD36 expression in response to 0.5 mM oleic acid:BSA treatment, which has been reported to redistribute CatB from the lysosome to the cytosol, where the majority of cellular LFABP is localized. Genetic knock-down of CatB in McA-RH7777 cells resulted in increased VLDL secretion as measured by 3H TAG DPM counting and immunoblot for ApoB in cell culture media, due to increased expression of LFABP and CD36 and increased FFA uptake. Knock-down of CatB also resulted in decreased cellular TAG as measured by 3H DPM counting due to increased VLDL secretion. CatB over-expression in McA-RH7777 cells resulted in decreased FFA uptake leading to decreased VLDL secretion, which was due to increased cleavage of LFABP. Co-localization of LFABP and CatB was observed exclusively under conditions of 0.5 mM oleic acid:BSA treatment. Based on these results, we can conclude that CatB plays a distinct physiological role in the turnover of LFABP and CD36 protein, which leads to suppressed uptake of FFA, and thus, reduced TAG synthesis and VLDL secretion.
Show less - Date Issued
- 2017
- Identifier
- CFE0006669, ucf:51236
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006669
- Title
- Membrane topology of a broad-spectrum resistance factor responsible for lipid modification in Enterococcus faecium.
- Creator
-
Harrison, Jesse, Roy, Herve, Teter, Kenneth, Phanstiel, Otto, University of Central Florida
- Abstract / Description
-
Aminoacylphosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that use aminoacyl-tRNAs as substrates to catalyze the addition of amino acids to phosphatidylglycerol (PG) in the cytoplasmic membranes of bacteria. Addition of amino acids to PG decreases the net negative charge of the membrane, conferring resistance to various classes of antibacterial agents (i.e., cationic antimicrobial peptides, beta-lactams, glycopeptides, and lipopeptides) and protecting the cell against...
Show moreAminoacylphosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that use aminoacyl-tRNAs as substrates to catalyze the addition of amino acids to phosphatidylglycerol (PG) in the cytoplasmic membranes of bacteria. Addition of amino acids to PG decreases the net negative charge of the membrane, conferring resistance to various classes of antibacterial agents (i.e., cationic antimicrobial peptides, beta-lactams, glycopeptides, and lipopeptides) and protecting the cell against osmotic stress and acidic conditions. aaPGS homologs are found in a variety of clinically relevant microorganisms, including Enterococcus faecium, which is increasingly found to be the etiologic agent of antibiotic-resistant nosocomial infections. Although the broad distribution of these virulence factors across bacterial species makes them attractive targets for therapeutic strategies, little is known about the structure of aaPGSs. Two aaPGS paralogs are found in E. faecium, one of which exhibits relaxed substrate specificity and is responsible for the transfer of Arg (R), Ala (A), and Lys (K) to PG (RakPGS). The catalytic site of RakPGS is located in the hydrophilic C-terminal domain, which is localized in the cytoplasm. The N-terminus contains an integral membrane domain that is thought to harbor flippase activity that translocates the neosynthesized aa-PG from the inner to the outer leaflet of the membrane. We are currently developing the substituted cysteine accessibility method (SCAM) and a dual-reporter fusion system, which exploits alkaline phosphatase (Pho) and ?-galactosidase (LacZ) activities, for investigating the membrane topology of RakPGS in E. faecium.
Show less - Date Issued
- 2015
- Identifier
- CFE0006318, ucf:51566
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006318
- Title
- The development of motuporamine derivatives and an investigation into their biological properties.
- Creator
-
Skruber, Kristen, Phanstiel, Otto, Teter, Kenneth, Vonkalm, Laurence, University of Central Florida
- Abstract / Description
-
This project investigates the synthesis of a class of compounds derived from a marine-based natural product and probes how iterative changes to its structure affect its derivatives' biological efficacy. The compound class of interest are the motuporamines which were isolated from the sea sponge Xestospongia exigua collected off the coast of Motupore island in Papua, New Guinea. The compounds for this project are predicated upon dihydromotuporamine C (Motu33), the compound that has been shown...
Show moreThis project investigates the synthesis of a class of compounds derived from a marine-based natural product and probes how iterative changes to its structure affect its derivatives' biological efficacy. The compound class of interest are the motuporamines which were isolated from the sea sponge Xestospongia exigua collected off the coast of Motupore island in Papua, New Guinea. The compounds for this project are predicated upon dihydromotuporamine C (Motu33), the compound that has been shown to be both cytotoxic to MDA-MB231 breast carcinoma cells and has antimetastatic efficacy. The motuporamine scaffold contains a large fifteen-membered saturated macrocycle and an appended polyamine component. A series of Motu33 derivatives were synthesized and evaluated for their ability to target the polyamine transport system as well as inhibit cell migration of human pancreatic cancer cells in vitro. By altering the polyamine component of the system we attempted to build smart antimetastatic compounds which target the upregulated polyamine transport system of human pancreatic cancers and block their migration.
Show less - Date Issued
- 2016
- Identifier
- CFE0006505, ucf:51390
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006505
- Title
- Pyroglutamylated amyloid beta peptides enhance non-fibrillogenic aggregation of the unmodified peptide.
- Creator
-
Goldblatt, Gregory, Tatulian, Suren, Chen, Bo, Teter, Kenneth, King, Stephen, University of Central Florida
- Abstract / Description
-
Alzheimer's disease (AD) is accompanied by abnormal extracellular deposition of amyloid b (Ab) peptide. This has led to the amyloid cascade hypothesis, causatively relating Ab with AD. While Ab deposits assume a fibrillar cross-b structure, prefibrillar oligomers of Ab have been identified as the main cytotoxic agents in AD. Pyroglutamylated amyloid beta (AbpE) peptides are N-terminally truncated and pyroglutamylated (at Glu3 or Glu11) Ab molecules that display enhanced cytotoxicity and...
Show moreAlzheimer's disease (AD) is accompanied by abnormal extracellular deposition of amyloid b (Ab) peptide. This has led to the amyloid cascade hypothesis, causatively relating Ab with AD. While Ab deposits assume a fibrillar cross-b structure, prefibrillar oligomers of Ab have been identified as the main cytotoxic agents in AD. Pyroglutamylated amyloid beta (AbpE) peptides are N-terminally truncated and pyroglutamylated (at Glu3 or Glu11) Ab molecules that display enhanced cytotoxicity and represent up to 50% of total Ab in AD brains. AbpE significantly enhances the toxicity of unmodified Ab by an unknown mechanism. Although in situ Ab populations are heterogeneous, the majority of studies have been conducted on single Ab species. Here, we examined the structural and morphological changes that occur in mixed Ab/AbpE samples. Circular dichroism and transmission electron microscopy data indicate that AbpE3-42 forms b-sheet structure and undergoes delayed fibrillogenesis compared to unmodified Ab1-42. Further, AbpE3-42 decelerates b-sheet formation in mixed Ab1-42/AbpE3-42 samples. FTIR measurements, using 13C-labeled Ab1-42 and unlabeled AbpE3-42, indicate that AbpE3-42 inhibits cross-b-sheet formation by Ab1-42, which explains the retardation of fibrillogenesis. FTIR on peptides 13C-labeled at specific segments provided site specific structural information. Based on these data, the monomeric Ab structure has been modeled as a b-hairpin stabilized by intramolecular H-bonding with an N-terminal a-helix. These hairpins likely form higher order aggregates through ionic and hydrophobic interactions between the C-terminus of one hairpin and the N-terminus of another. Utilizing a novel technique, hydration from gas phase, we examined the a-helix to b-sheet transitions of these peptides. When combined, AbpE3-42 and Ab1-42 mutually inhibit intermolecular b-sheet formation, instead promoting formation of AbpE3-42/Ab1-42 hetero-oligomers of intramolecular H-bonding. These hetero-oligomers displayed enhanced toxicity to PC12 cells compared to individual peptides and induced greater calcium release from lipid vesicles than unmodified Ab. These results indicate that Ab and AbpE mutually inhibit fibrillogenesis and stabilize hetero-oligomers of enhanced cytotoxicity, possibly through a membrane permeabilization mechanism. Collectively, our findings lead to a new concept that Ab/AbpE hetero-oligomers, not just Ab or AbpE oligomers, are the main cytotoxic species in AD
Show less - Date Issued
- 2016
- Identifier
- CFE0006108, ucf:51195
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006108
- Title
- The Anti-toxin Properties of Grape Seed Phenolic Compounds.
- Creator
-
Cherubin, Patrick, Teter, Kenneth, Zervos, Antonis, Roy, Herve, Phanstiel, Otto, University of Central Florida
- Abstract / Description
-
Corynebacterium diphtheriae, Pseudomonas aeruginosa, Ricinus communis, Shigella dysentariae, and Vibrio cholerae produce AB toxins which share the same basic structural characteristics: a catalytic A subunit attached to a cell-binding B subunit. All AB toxins have cytosolic targets despite an initial extracellular location. AB toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against these toxins are therefore hard to...
Show moreCorynebacterium diphtheriae, Pseudomonas aeruginosa, Ricinus communis, Shigella dysentariae, and Vibrio cholerae produce AB toxins which share the same basic structural characteristics: a catalytic A subunit attached to a cell-binding B subunit. All AB toxins have cytosolic targets despite an initial extracellular location. AB toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against these toxins are therefore hard to develop because they use different surface receptors, entry mechanisms, enzyme activities, and cytosolic targets.We have found that grape seed extract provides resistance to five different AB toxins: diphtheria toxin (DT), P. aeruginosa exotoxin A (ETA), ricin, Shiga toxin, and cholera toxin (CT). To identify individual compounds in grape seed extract that are capable of inhibiting the activities of these AB toxins, we screened twenty common phenolic compounds of grape seed extract for anti-toxin properties. Three compounds inhibited DT, four inhibited ETA, one inhibited ricin, and twelve inhibited CT. Additional studies were performed to determine the mechanism of inhibition against CT. Two compounds inhibited CT binding to the cell surface and even stripped bound CT off the plasma membrane of a target cell. Two other compounds inhibited the enzymatic activity of CT. We have thus identified individual toxin inhibitors from grape seed extract and some of their mechanisms of inhibition against CT. This work will help to formulate a defined mixture of phenolic compounds that could potentially be used as a therapeutic against a broad range of AB toxins.
Show less - Date Issued
- 2014
- Identifier
- CFE0005315, ucf:50510
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005315
- Title
- Modulation of cholera toxin structure and function by host proteins.
- Creator
-
Burress, Helen, Teter, Kenneth, Self, William, Zervos, Antonis, Tatulian, Suren, University of Central Florida
- Abstract / Description
-
Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the...
Show moreCholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared (FTIR) 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 its refolding of CTA1 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance (SPR) 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 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. This activity is consistent with its role as a chaperone that refolds endogenous cytosolic proteins as part of a foldosome complex consisting of Hsp90, Hop, Hsp40, p23, and Hsc70. A role for Hsc70 in CT intoxication has not yet been established. Here, biophysical, biochemical, and cell-based assays demonstrate Hsp90 and Hsc70 play overlapping roles in the processing of CTA1. Using SPR we determined that Hsp90 and Hsc70 could bind independently to CTA1 at distinct locations with high affinity, even in the absence of the Hop linker. Studies using isotope-edited FTIR spectroscopy found that, like Hsp90, Hsc70 induces a gain-of-structure in unfolded CTA1. The interaction between CTA1 and Hsc70 is essential for intoxication, as an RNAi-induced loss of the Hsc70 protein generates a toxin-resistant phenotype. Further analysis using isotope-edited FTIR spectroscopy demonstrated that the addition of both Hsc70 and Hsp90 to unfolded CTA1 produced a gain-of-structure above that of the individual chaperones. Our data suggest that CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with extraction from the ER. The subsequent binding of Hsc70 further refolds CTA1 in a manner not previously observed in foldosome complex formation. The interaction of CTA1 with these chaperones is essential to intoxication and this work elucidates details of the intoxication process not previously known.
Show less - Date Issued
- 2014
- Identifier
- CFE0005310, ucf:50511
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005310
