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(1 - 16 of 16)
- 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
- ATP-Induced Disassembly of CDTB/CDTC Heterodimer of Cytolethal Distending Toxin.
- Creator
-
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
- IDENTIFICATION OF THE DOMAIN(S) IN PROTEIN DISULFIDE ISOMERASE REQUIRED FOR BINDING AND DISASSEMBLY OF THE CHOLERA HOLOTOXIN.
- Creator
-
Herndon, Laura, Teter, Ken, University of Central Florida
- Abstract / Description
-
Cholera, caused by the secretion of cholera toxin (CT) by Vibrio cholerae within the intestinal lumen, triggers massive secretory diarrhea which may lead to life-threatening dehydration. CT is an AB5-type protein toxin that is comprised of an enzymatically active A1 chain, an A2 linker, and a cell-binding B pentamer. Once secreted, the CT holotoxin moves from the cell surface to the endoplasmic reticulum (ER) of a host target cell. To cause intoxication, CTA1 must be displaced from CTA2/CTB5...
Show moreCholera, caused by the secretion of cholera toxin (CT) by Vibrio cholerae within the intestinal lumen, triggers massive secretory diarrhea which may lead to life-threatening dehydration. CT is an AB5-type protein toxin that is comprised of an enzymatically active A1 chain, an A2 linker, and a cell-binding B pentamer. Once secreted, the CT holotoxin moves from the cell surface to the endoplasmic reticulum (ER) of a host target cell. To cause intoxication, CTA1 must be displaced from CTA2/CTB5 in the ER and is then transferred to the cytosol where it induces a diarrheal response by stimulating the efflux of chloride ions into the intestinal lumen. Protein disulfide isomerase (PDI), a resident ER oxidoreductase and chaperone, is involved in detaching CTA1 from the holotoxin. The PDI domain(s) that binds to CTA1 and precisely how this interaction is involved in CTA1 dissociation from the holotoxin are unknown. The goal of this project is to identify which domain(s) of PDI is responsible for binding to and dislodging CTA1 from the CT holotoxin. Through incorporation of ELISA, surface plasmon resonance (SPR), and Fourier transform infrared (FTIR) spectroscopy techniques in conjunction with a panel of purified PDI deletion constructs, this project aims to provide important molecular insight into a crucial interaction of the CT intoxication process.
Show less - Date Issued
- 2015
- Identifier
- CFH0004792, ucf:45334
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004792
- 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
- TRANSLOCATION OF THE CHOLERA TOXIN A1 SUBUNIT FROM THE ENDOPLASMIC RETICULUM TO THE CYTOSOL.
- Creator
-
Taylor, Michael, Teter, Ken, University of Central Florida
- Abstract / Description
-
AB-type protein toxins such as cholera toxin (CT) consist of a catalytic A subunit and a cell-binding B subunit. CT proceeds through the secretory pathway in reverse, termed retrograde trafficking, and is delivered to the endoplasmic reticulum (ER). In order for the catalytic A1 subunit to become active it must separate from the rest of the holotoxin, and this dissociation event occurs in the ER lumen. CTA1 assumes an unfolded conformation upon dissociation from the holotoxin and is...
Show moreAB-type protein toxins such as cholera toxin (CT) consist of a catalytic A subunit and a cell-binding B subunit. CT proceeds through the secretory pathway in reverse, termed retrograde trafficking, and is delivered to the endoplasmic reticulum (ER). In order for the catalytic A1 subunit to become active it must separate from the rest of the holotoxin, and this dissociation event occurs in the ER lumen. CTA1 assumes an unfolded conformation upon dissociation from the holotoxin and is recognized by ER- associated degradation (ERAD), a quality control system that recognizes and exports misfolded proteins to the cytosol for degradation by the 26S proteasome. CTA1 is not degraded by the 26S proteasome because it has few sites for poly-ubitiquination, which is recognized by the cap of the 26S proteasome for degradation. Thus, CTA1 escapes the degradation of ERAD while at the same time using it as a transport mechanism into the cytosol. It was originally proposed that CTA1 is thermally stable and that ER chaperones actively unfolded CTA1 for translocation to the cytosol. In contrast, we hypothesized that the dissociated CTA1 subunit would unfold spontaneously at 37°C. This study focused on the three conditions linked to CTA1 instability and translocation: (i) CTA1 dissociation from the holotoxin, (ii) the translocation-competent conformation of CTA1, and (iii) the extraction of CTA1 from the ER into the cytosol. Disruption of any of these events will confer resistance to the toxin. The original model suggested that PDI actively unfolds CTA1 to allow for translocation. However, Fourier transform infrared spectroscopy (FTIR) and surface plasmon resonance (SPR) data we have gathered demonstrated that PDI dislodges CTA1 from the rest of the holotoxin without unfolding CTA1. Once released by the holotoxin, CTA1 spontaneously unfolds. PDI is thus required for the toxicity of CT, but not as an unfoldase as originally proposed. CTA1 must maintain an unfolded conformation to keep its translocation-competent state. Based on our model, if CTA1 is stabilized then it will not be able to activate the ERAD translocation system. Our SPR and toxicity results demonstrated that treatment with 4-phenylbutyrate (PBA), a chemical chaperone, stabilizes the structure of CTA1. This stabilization resulted in a decrease in translocation from the ER to the cytosol and a block of intoxication, which makes it a viable candidate for a therapeutic. Because CTA1 exits the ER in an unfolded state, there must be a driving force for this translocation. We hypothesized that Hsp90, a cytosolic chaperone, is responsible for the translocation of CTA1 across the membrane. Previous research had shown Hsp90 to be present on the cytosolic face of the ER and had also shown that Hsp90 will refold exogenously added proteins that enter the cytosol. Using drug treatments and RNAi, we found that Hsp90 is required for the translocation of CTA1 from the ER lumen to the cytosol, a brand new function for this chaperone. We have provided evidence to support a new, substantially different model of CTA1 translocation. CTA1 does not masquerade as a misfolded protein in order to utilize ERAD for entry into the cytosol; it actually becomes misfolded and is treated as any other ERAD substrate. The spontaneous unfolding of CTA1 is the key to its recognition by ERAD and ultimately its translocation into the cytosol. Host factors play very important roles in intoxication by AB toxins and are targets for blocking intoxication.
Show less - Date Issued
- 2011
- Identifier
- CFE0003733, ucf:48784
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003733
- Title
- The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin.
- Creator
-
Bader, Carly, Teter, Kenneth, Zervos, Antonis, Jewett, Travis, Tatulian, Suren, University of Central Florida
- Abstract / Description
-
Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much...
Show moreCholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the Golgi apparatus and then to the ER. Here the A1 subunit detaches from the rest of the toxin and enters the cytosol. The disordered conformation of free CTA1 facilitates toxin export to the cytosol by activating a quality control mechanism known as ER-associated degradation. The return to a folded structure in the cytosol allows CTA1 to attain an active conformation for modification of its Gs? target through ADP-ribosylation. This modification locks the protein in an active state which stimulates adenylate cyclase and leads to elevated levels of cAMP. A chloride channel located in the apical enterocyte membrane opens in response to signaling events induced by these elevated cAMP levels. The osmotic movement of water into the intestinal lumen that results from the chloride efflux produces the characteristic profuse watery diarrhea that is seen in intoxicated individuals.The current model of intoxication proposes only one molecule of cytosolic toxin is required to affect host cells, making therapeutic treatment nearly impossible. However, based on emerging evidence, we hypothesize a threshold quantity of toxin must be present within the cytosol of the target cell in order to elicit a cytopathic effect. Using the method of surface plasmon resonance along with toxicity assays, I have, for the first time, directly measured the efficiency of toxin delivery to the cytosol and correlated the levels of cytosolic toxin to toxin activity. I have shown CTA1 delivery from the cell surface to the cytosol is an inefficient process with only 2.3 % of the surface bound CTA1 appearing in the cytosol after 2 hours of intoxication. I have also determined and a cytosolic quantity of more than approximately .05ng of cytosolic CTA1 must be reached in order to elicit a cytopathic effect. Furthermore, CTA1 must be continually delivered from the cell surface to the cytosol in order to overcome the constant proteasome-mediated clearance of cytosolic toxin. When toxin delivery to the cytosol was blocked, this allowed the host cell to de-activate Gs?, lower cAMP levels, and recover from intoxication. Our work thus indicates it is possible to treat cholera even after the onset of disease. These findings challenge the idea of irreversible cellular toxicity and open the possibility of post-intoxication treatment options.
Show less - Date Issued
- 2013
- Identifier
- CFE0004810, ucf:49759
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004810
- 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
- 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
- EXPRESSION OF HETEROLOGOUS PROTEINS IN TRANSGENIC TOBACCO CHLOROPLASTS TO PRODUCE A BIOPHARMACEUTICAL AND BIOPOLYMER.
- Creator
-
Devine, Andrew, Daniell, Henry, University of Central Florida
- Abstract / Description
-
The chloroplast has been demonstrated to be an ideal compartment to accumulate certain proteins or their biosynthetic products that would be harmful if they were accumulated in the cytoplasm. Hyper-expression of foreign proteins in chloroplast transgenics has accumulated up to 46% total soluble protein, this is possible due to the ~100 chloroplast genomes per chloroplast and ~100 chloroplasts per cell which can therefore, contain up to 10,000 copies of the transgene. Maternal gene inheritance...
Show moreThe chloroplast has been demonstrated to be an ideal compartment to accumulate certain proteins or their biosynthetic products that would be harmful if they were accumulated in the cytoplasm. Hyper-expression of foreign proteins in chloroplast transgenics has accumulated up to 46% total soluble protein, this is possible due to the ~100 chloroplast genomes per chloroplast and ~100 chloroplasts per cell which can therefore, contain up to 10,000 copies of the transgene. Maternal gene inheritance of plastids in most crop plants results in natural gene containment. Chloroplast transformation also eliminates positional effects that are frequently observed with nuclear transformation and no gene silencing has been observed so far at the level of transcription or translation. Consequently, independent chloroplast transgenic lines have very similar levels of foreign gene expression and there is no need to screen hundreds of transgenic events. The chloroplast genome has also been used in molecular farming to express human therapeutic proteins, vaccines for human or animal use and biomaterials. In this study we have produced a Nicotiana tabacum cv. petit Havana chloroplast transgenic line that expresses a cholera toxin B subunit (from Vibrio Cholerae)-human proinsulin (a,b and c chain) fusion protein, designated CTB-Pris. The pLD-PW vector contains the CTB-Pris gene cloned into the universal chloroplast transformation vector pLD-ctv in which the 16S rRNA promoter drives the aadA gene selectable marker, which confers resistance to spectinomycin; the psbA 5' untranslated region (UTR) which enhances translation of CTB-Pris in the presence of light and the psbA 3'UTR confers transcript stability. The trnI and trnA homologous flanking sequences facilitated site-specific integration of transgenes into the tobacco chloroplast genome. Site-specific integration was demonstrated by PCR and Southern blot analysis with probes for CTB-Pris. Western Blot analysis has demonstrated the presence of abundant CTB-Pris in transgenic plants with both CTB polyclonal and proinsulin monoclonal antibodies. Southern blot analysis has also confirmed that homoplasmy had been achieved in the T0 generation. The expression levels for CTB-Proinsulin varied between 270ìg/100mg to 364.8ìg/100mg of plant tissue which equates to ~30% total soluble protein. In the second study the E. coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated in the tobacco chloroplast genome under the control of the light-regulated psbA 5' untranslated region. CPL catalyzes the direct conversion of chorismate – an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids – to pHBA and pyruvate. pHBA is the major monomer in liquid crystal polymers (LCPs). These thermotropic polyesters have excellent properties, including high strength/stiffness, low melt viscosity, property retention at elevated temperatures, environmental resistance and low gas permeability. The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13-18% DW, while the oldest leaves had levels as high as 26.5% DW. The highest CPL enzyme activity observed in total leaf material was 50,783 pkat/mg of protein, which is equivalent to ~35% of the total soluble protein. Animal studies in the Daniell lab, suggest that the CTB-Proinsulin producing plants suppress insulitis and clinical symptoms of diabetes in NOD mice. These observations demonstrate the versatility of chloroplast gene expression for production of biopharmaceuticals and biopolymers.
Show less - Date Issued
- 2006
- Identifier
- CFE0001056, ucf:46794
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001056
- 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
- ROLE OF MEMBRANE LIPIDS IN MODULATING PROTEIN STRUCTURE & FUNCTION.
- Creator
-
Ray, Supriyo, Tatulian, Suren, University of Central Florida
- Abstract / Description
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A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated...
Show moreA-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A2 (PLA2). Lipid thermal phase transition was found to have a dramatic effect on PLA2 activity. It was also established that although membrane binding and insertion was essential for of PLA2 activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA2 secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA2. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.
Show less - Date Issued
- 2011
- Identifier
- CFE0004035, ucf:49184
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004035
- Title
- Expression and functional evaluation of exendin 4 fused to cholera toxin B subunit in tobacco chloroplasts to treat type 2 diabetes.
- Creator
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Nityanandam, Ramya, Daniell, Henry, Naser, Saleh, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
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The prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4)...
Show moreThe prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4), lizard derived GLP-1R agonist, was expressed as cholera toxin B subunit (CTB)-fusion protein in chloroplasts of tobacco to facilitate transmucosal delivery in the gut by utilizing the ability of CTB pentamer to bind the GM1 receptors on the intestinal epithelium and to bioencapsulate EX4 within plant cells to confer protection in the digestive system. The LAMD tobacco leaves were bombarded with chloroplast vectors expressing modified EX4. The transgene integration was confirmed by PCR analysis and Southern blot analysis. Densitometric analysis revealed expression level of the protein varied from 9-13% of the total leaf protein depending on the developmental stage and time of harvest. The pentameric structure and functionality of CTB-EX4 fusion protein was confirmed by CTB-GM1 binding assay. The effect of transplastomic protein on insulin secretion was tested in ?-TC6, a mouse pancreatic cell line. The plant derived CTB-EX4, partially purified with anti-CTB antibody conjugated protein A beads, showed the increase of insulin ~ 2.5 fold increase when compared to untreated cells. The transplastomic protein showed a linear increase in insulin secretion comparable to the commercially available EX4. The current cost of treatment with EX4 varies between $1800-$2200, annually. Production of functional EX4 in plants should facilitate low cost orally deliverable form of this drug for treatment of type 2 diabetes.
Show less - Date Issued
- 2011
- Identifier
- CFE0004485, ucf:49306
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004485
- 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
- Modulation of cholera toxin structure and function by host proteins.
- Creator
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Burress, Helen, Teter, Kenneth, Self, William, Zervos, Antonis, Tatulian, Suren, University of Central Florida
- Abstract / Description
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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
- Title
- AMYLOID-BETA42 TOXICITY REDUCTION IN HUMAN NEUROBLASTOMA CELLS USING CHOLERA TOXIN B SUBUNIT-MYELIN BASIC PROTEIN EXPRESSED IN CHLOROPLASTS.
- Creator
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Ayache, Alexandra, Daniell, Henry, University of Central Florida
- Abstract / Description
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Alzheimer's disease (AD) is an age progressive neurodegenerative brain disorder, affecting 37 million people worldwide. Cleavage of amyloid precursor protein by β- and γ-secretase produces the amyloid-beta (Aβ) protein, which significantly contributes to AD pathogenesis. The Aβ aggregates, formed at the surface of neurons and intracellularly, cause neurotoxicity and decrease synaptic function. Inhibiting or degrading Aβ accumulation is a key goal for development of new AD treatments. Evidence...
Show moreAlzheimer's disease (AD) is an age progressive neurodegenerative brain disorder, affecting 37 million people worldwide. Cleavage of amyloid precursor protein by β- and γ-secretase produces the amyloid-beta (Aβ) protein, which significantly contributes to AD pathogenesis. The Aβ aggregates, formed at the surface of neurons and intracellularly, cause neurotoxicity and decrease synaptic function. Inhibiting or degrading Aβ accumulation is a key goal for development of new AD treatments. Evidence shows that human Myelin Basic Protein (MBP) binds to and degrades Aβ thereby, preventing cytotoxicity. A potential method for oral drug delivery that will allow plant-derived bioencapsulated MBP to pass through intestinal epithelium and bypass denaturing stomach acidity is quite novel. Cholera Toxin B subunit (CTB), when fused with MBP, can serve as a vehicle for oral delivery of this chloroplast expressed therapeutic protein into the systemic circulation. Within chloroplast, CTB forms a pentameric structure that binds to GM1 ganglioside receptors, allowing receptor-mediated endocytosis. In order to investigate protein entry through neuronal GM1 receptors, we first created CTB fused to the green fluorescent protein (GFP). Incubation of this fusion protein with human neuroblastoma cells resulted in GFP entry into these cells whereas GFP alone was unable to enter. Similarly, co-incubation of CTB-MBP, via neuronal GM1 binding, allowed MBP to reduce neurotoxicity of Aβ42 treated cells by 37.1%. Delivery of CTB-MBP through GM1 receptor mediated binding should therefore facilitate oral administration, storage, heat stability and low cost AD treatment.
Show less - Date Issued
- 2012
- Identifier
- CFH0004249, ucf:44916
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004249
- Title
- Evolution and distribution of phenotypic diversity in the venom of Mojave Rattlesnakes (Crotalus scutulatus).
- Creator
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Strickland, Jason, Savage, Anna, Parkinson, Christopher, Hoffman, Eric, Rokyta, Darin, University of Central Florida
- Abstract / Description
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Intraspecific phenotype diversity allows for local adaption and the ability for species to respond to changing environmental conditions, enhancing survivability. Phenotypic variation could be stochastic, genetically based, and/or the result of different environmental conditions. Mojave Rattlesnakes, Crotalus scutulatus, are known to have high intraspecific venom variation, but the geographic extent of the variation and factors influencing venom evolution are poorly understood. Three primary...
Show moreIntraspecific phenotype diversity allows for local adaption and the ability for species to respond to changing environmental conditions, enhancing survivability. Phenotypic variation could be stochastic, genetically based, and/or the result of different environmental conditions. Mojave Rattlesnakes, Crotalus scutulatus, are known to have high intraspecific venom variation, but the geographic extent of the variation and factors influencing venom evolution are poorly understood. Three primary venom types have been described in this species based on the presence (Type A) or absence (Type B) of a neurotoxic phospholipase A2 called Mojave toxin and an inverse relationship with the presence of snake venom metalloproteinases (SVMPs). Individuals that contain both Mojave toxin and SVMPs, although rare, are the third, and designated Type A + B. I sought to describe the proteomic and transcriptomic venom diversity of C. scutulatus across its range and test whether diversity was correlated with genetic or environmental differences. This study includes the highest geographic sampling of Mojave Rattlesnakes and includes the most venom-gland transcriptomes known for one species. Of the four mitochondrial lineages known, only one was monophyletic for venom type. Environmental variables poorly correlated with the phenotypes. Variability in toxin and toxin family composition of venom transcriptomes was largely due to differences in transcript expression. Four of 19 toxin families identified in C. scutulatus account for the majority of differences in toxin number and expression variation. I was able to determine that the toxins primarily responsible for venom types are inherited in a Mendelian fashion and that toxin expression is additive when comparing heterozygotes and homozygotes. Using the genetics to define venom type is more informative and the Type A + B phenotype is not unique, but rather heterozygous for the PLA2 and/or SVMP alleles. Intraspecific venom variation in C. scutulatus highlights the need for fine scale ecological and natural history information to understand how phenotypic diversity is generated and maintained geographically through time.
Show less - Date Issued
- 2018
- Identifier
- CFE0007252, ucf:52198
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007252
